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1
McDonnell, Mollie M., Suzanne C. Karvonen, Amit Gaba, Ben Flath, Linda Chelico, and Michael Emerman. "Highly-potent, synthetic APOBEC3s restrict HIV-1 through deamination-independent mechanisms." PLOS Pathogens 17, no. 6 (June 25, 2021): e1009523. http://dx.doi.org/10.1371/journal.ppat.1009523.
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The APOBEC3 (A3) genes encode cytidine deaminase proteins with potent antiviral and anti-retroelement activity. This locus is characterized by duplication, recombination, and deletion events that gave rise to the seven A3s found in primates. These include three single deaminase domain A3s (A3A, A3C, and A3H) and four double deaminase domain A3s (A3B, A3D, A3F, and A3G). The most potent of the A3 proteins against HIV-1 is A3G. However, it is not clear if double deaminase domain A3s have a generalized functional advantage to restrict HIV-1. In order to test whether superior restriction factors could be created by genetically linking single A3 domains into synthetic double domains, we linked A3C and A3H single domains in novel combinations. We found that A3C/A3H double domains acquired enhanced antiviral activity that is at least as potent, if not better than, A3G. Although these synthetic double domain A3s package into budding virions more efficiently than their respective single domains, this does not fully explain their gain of antiviral potency. The antiviral activity is conferred both by cytidine-deaminase dependent and independent mechanisms, with the latter correlating to an increase in RNA binding affinity. T cell lines expressing this A3C-A3H super restriction factor are able to control replicating HIV-1ΔVif infection to similar levels as A3G. Together, these data show that novel combinations of A3 domains are capable of gaining potent antiviral activity to levels similar to the most potent genome-encoded A3s, via a primarily non-catalytic mechanism.
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Takei, Hisashi, Masanori Fujii, Sohei Nakayama, Ikei Kobayashi, Keisuke Shindo, Kotaro Shirakawa, Akifumi Takaori-Kondo, and Susumu Kobayashi. "Alternative Splicing of APOBEC3D Generates Functional Diversity and Its Role As a DNA Mutator." Blood 128, no. 22 (December 2, 2016): 5107. http://dx.doi.org/10.1182/blood.v128.22.5107.5107.
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Abstract Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family consists of 11 members: APOBEC1, APOBEC2, seven APOBEC3s (A3s) (A/B/C/D/F/G/H), APOBEC4, and the activation-induced deaminase (AID). APOBEC1, A3s, and AID have cytidine deaminase activity and induce a cytidine (C) to thymidine (T) transition. The main function of A3s is to trigger an innate immune response to viral infection such as human immunodeficiency virus-1. Recently, it was reported that several APOBEC family proteins can induce somatic mutations into genomic DNA and thus promote cancer development. For example, AID-mediated somatic mutations contribute to B-cell lymphoma and A3B can be an enzymatic source in solid tumors such as breast cancer. However, little is known about other A3 member proteins. To determine the expression of A3 in hematopoietic cells, we performed quantitative PCR in leukemia cell lines. A3B, A3C, A3D, A3F and A3G were detected in all cell lines, but not A3A or A3H. Next, we analyzed published The Cancer Genome Atlas data from patients with acute myeloid leukemia to find somatic alterations in A3 genes using cBioPortal (http://www.cbioportal.org/). We found that each A3 was upregulated at 4-7 % of all samples and the total frequency of 23 %. Interestingly, overall survival of patients with A3upregulation was lower than those without upregulation, suggesting an important role of A3s in pathogenesis of leukemia. As it has been shown that A3A, A3B, and A3D show capacity to inflict DNA damage, we decided to investigate whether A3D can induce mutations in foreign and genomic DNA. During our attempt to generate A3D expression constructs, we detected multiple bands in leukemia and lung cancer cell lines. Based on our analysis and previous reports, we confirmed that there are at least 7 transcript variants (v1 to 7). Because A3D v3, v4 and v5 lack cytidine deaminase domains, we decided to examine A3Dv1, v2, v6 and v7. To determine whether A3D variants have foreign DNA editing activity, we performed differential DNA denaturation (3D)-PCR in HEK293T cells co-transfected with expression vectors for EGFP, UNG inhibitor, and pCAG-GS containing A3Dv1, v2, v6, v7, A3B or empty control. Total DNA was isolated 4 days after transfection. PCR products were detected at lower denaturation temperature (Td) in cells expressing A3B and all A3D variants vectors compared to control. Sequences of PCR products at lowest Td revealed that mutation frequencies in EGFP gene were 6.1-10.5 per 103 bps in cells transfected with all A3D variants and 18.7 per 103bps in cells transfected with A3B. Of note, 90 % of mutations were C/G to T/A transition in cells transfected with A3B, but in cells transfected with A3D these were 30-60 %. Next we performed 3D-PCR in HEK293T cells retrovirally infected with MigR1-IRES-EGFP containing A3Dv1, v2, v6, v7, A3B or empty to determine genomic DNA editing activity. Total DNA was isolated 4 weeks after infection. At lower Td, PCR products were detected only in cells expressing A3Dv1 and A3B. PCR products in cells expressing A3Dv2, v6, v7 were detected only at same Td as control. Sequences of the PCR products at lowest Td revealed that the mutation frequency in the EGFP gene was 22.2 per 103 bps in cells expressing A3Dv1, and 56.9 per 103 bps in cells expressing A3B. However mutation frequencies in cells expressing A3Dv6 or v7 were lower than 2 per 103bps and no mutations were detected in cells expressing A3Dv2. All of these mutations were C/G to T/A transition. Interestingly, we found 4 clones with deletion in cells expressing A3Dv1, v6 and A3B. In addition, all clones had several bases of micro homologous sequences in broken ends before joining. These results suggested that double strand breaks in genomic DNA were repaired by microhomology-mediated end joining. These DNA editing assays showed that all A3D variants had ability to induce mutations in foreign DNA, and A3Dv1 was major isoform which had ability to induce mutations in genomic DNA. Taken together, our experiments showed that A3D can be a DNA mutator and alternative splicing generates functional diversity of A3D. These findings suggest that A3D may contribute to development of hematopoietic malignancies. Disclosures Takaori-Kondo: Mochida Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Kyowa Kirin: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.
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Marin, Mariana, Sheetal Golem, Kristine M. Rose, Susan L. Kozak, and David Kabat. "Human Immunodeficiency Virus Type 1 Vif Functionally Interacts with Diverse APOBEC3 Cytidine Deaminases and Moves with Them between Cytoplasmic Sites of mRNA Metabolism." Journal of Virology 82, no. 2 (October 31, 2007): 987–98. http://dx.doi.org/10.1128/jvi.01078-07.
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ABSTRACT VifIIIB, which has been a standard model for the viral infectivity factor of human immunodeficiency virus type 1 (HIV-1), binds the cytidine deaminase APOBEC3G (A3G) and induces its degradation, thereby precluding its lethal incorporation into assembling virions. Additionally, VifIIIB less efficiently degrades A3F, another potent anti-HIV-1 cytidine deaminase. Although the APOBEC3 paralogs A3A, A3B, and A3C have weaker anti-HIV-1 activities and are only partially degraded by VifIIIB, we found that VifIIIB induces their emigration from the nucleus to the cytosol and thereby causes net increases in the cytosolic concentrations and anti-HIV-1 activities of A3A and A3B. In contrast, some other Vifs, exemplified by VifHXB2 and VifELI-1, much more efficiently degrade and thereby neutralize all APOBEC3s. Studies focused mainly on A3F imply that it occurs associated with mRNA-PABP1 in translationally active polysomes and to a lesser extent in mRNA processing bodies (P-bodies). A3F appears to stabilize the P-bodies with which it is associated. A correspondingly small proportion of VifIIIB also localizes in P-bodies in an A3F-dependent manner. Stress causes A3A, A3B, A3C, and A3F to colocalize efficiently with VifIIIB and mRNA-PABP1 complexes in stress granules in a manner that is prevented by cycloheximide, an inhibitor of translational elongation. Coimmunoprecipitation studies suggest that Vifs from different HIV-1 isolates associate with all tested APOBEC3s. Thus, Vifs interact closely with structurally diverse APOBEC3s, with effects on their subcellular localization, degradation rates, and antiviral activities. Cytosolic APOBEC3-Vif complexes are predominantly bound to mRNAs that dynamically move between translationally active and storage or processing pools.
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Vasudevan, Ananda Ayyappan Jaguva, Sander H. J. Smits, Astrid Höppner, Dieter Häussinger, Bernd W. Koenig, and Carsten Münk. "Structural features of antiviral DNA cytidine deaminases." Biological Chemistry 394, no. 11 (November 1, 2013): 1357–70. http://dx.doi.org/10.1515/hsz-2013-0165.
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Abstract The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five β sheets arranged in a characteristic motif (α1-β1-β2/2′-α2-β3-α3-β4-α4-β5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review.
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Talluri, Srikanth, Mehmet Kemal Samur, Leutz Buon, Stekla A. Megan, Purushothama Nanjappa, Rao Prabhala, Masood A. Shammas, and Nikhil C. Munshi. "Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma." Blood 128, no. 22 (December 2, 2016): 803. http://dx.doi.org/10.1182/blood.v128.22.803.803.
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Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.
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Pery, Erez, Kottampatty S. Rajendran, Andrew Jay Brazier, and Dana Gabuzda. "Regulation of APOBEC3 Proteins by a Novel YXXL Motif in Human Immunodeficiency Virus Type 1 Vif and Simian Immunodeficiency Virus SIVagm Vif." Journal of Virology 83, no. 5 (December 24, 2008): 2374–81. http://dx.doi.org/10.1128/jvi.01898-08.
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ABSTRACT The APOBEC3 cytidine deaminases are potent antiviral factors that restrict the replication of human immunodeficiency virus type 1 (HIV-1). In HIV-1-infected CD4+ T cells, the viral accessory protein Vif binds to APOBEC3G (A3G), APOBEC3F (A3F), and APOBEC3C (A3C) and targets these proteins for polyubiquitination by forming an E3 ubiquitin ligase with cullin 5. Previous studies identified regions of HIV-1 Vif, 40YRHHY44 and 12QVDRMR17, which are important for interaction with A3G and A3F, respectively, and showed that Vif residues 54 to 71 are sufficient for A3G binding. Here, we identify 69YXXL72 as a novel conserved motif in HIV-1 Vif that mediates binding to human A3G and its subsequent degradation. Studies on other APOBEC3 proteins revealed that Tyr69 and Leu72 are important for the degradation of A3F and A3C as well. Similar to A3F, A3C regulation is also mediated by Vif residues 12QVDRMR17. Simian immunodeficiency virus (SIV) Vif was shown to bind and degrade African green monkey A3G (agmA3G) and, unexpectedly, human A3C. The YXXL motif of SIVagm Vif was important for the inactivation of agmA3G and human A3C. Unlike HIV-1 Vif, however, SIVagm Vif does not require Tyr40 and His43 for agmA3G degradation. Tyr69 in the YXXL motif was critical for binding of recombinant glutathione S-transferase-Vif(1-94) to A3G in vitro. These results suggest that the YXXL motif in Vif is a potential target for small-molecule inhibitors to block Vif interaction with A3G, A3F, and A3C, and thereby protect cells against HIV-1 infection.
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Atchekzaï, Jean, Bernard Bonnetot, Jean-Claude Duplan, Bernard Fenet, Bernard Ferage, and Henri Mongeot. "Unprecedented example of a3J(13C,11B) through a CCNB linkage." Magnetic Resonance in Chemistry 27, no. 7 (July 1989): 699–701. http://dx.doi.org/10.1002/mrc.1260270719.
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Nakashima, Masaaki, Shingo Kitamura, Teppei Kurosawa, Hirotaka Ode, Takashi Kawamura, Mayumi Imahashi, Yoshiyuki Yokomaku, Nobuhisa Watanabe, Wataru Sugiura, and Yasumasa Iwatani. "Crystal structure of the Vif-interaction domain of the anti-viral APOBE3F." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C123. http://dx.doi.org/10.1107/s2053273314098763.
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Human cells express a family of cytidine deaminases, called APOBEC3 (A3) (A3A, B, C, D, F, G, and H). The family enzymes, especially A3G and A3F potentially inhibit replication of retroviruses including HIV-1. However, HIV-1 overcomes the A3-mediated antiviral system by expressing a virus-encoded antagonist, viral infectivity factor (Vif) protein. In HIV-1-infected cells, Vif specifically binds with A3 followed by proteasomal degradation of A3. Hence, inhibition of the interaction between A3 and Vif is an attractive strategy for developing novel anti-HIV-1 drugs. To date, we have determined the first crystal structure of A3 with Vif-binding interface, A3C (PDB ID: 3VOW). In addition, our extensive mutational analysis, based on the A3C structure, revealed that structural features of the Vif-binding interface are highly conserved among A3C, DE, and F [1]. However, more recently, Bohn et al. and Karen et al. have shown the crystal structures of mutant A3F C-terminal domain (CTD) which is responsible for the Vif interaction, and have predicted more extended area, including our identified residues, for the interface on the A3F CTD [2][3]. To clarify the Vif-binding interface of A3F, we sought to determine the crystal structure of the wild-type A3F CTD and evaluated contributions of the additional residues for the Vif-interaction interface by virological method. First, we have successfully determined the crystal structure of A3F CTD at 2.75 Å resolution. Furthermore, we have identified four additional residues unique on the A3F CTD but not A3C for Vif interaction, which are located in the vicinity of our previously reported interface. These results demonstrated that the structural features of Vif-binding interface are indeed conserved between A3C and A3F. Taken together, these results will provide the fine-tuned structure information to understand the binding between A3 and Vif and to facilitate a development of novel anti-HIV-1 compounds targeting A3 proteins.
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Wang, Feng-xiang, Jialing Huang, Hangxiang Zhang, Xinliang Ma, and Hui Zhang. "APOBEC3G upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells." Journal of General Virology 89, no. 3 (March 1, 2008): 722–30. http://dx.doi.org/10.1099/vir.0.83530-0.
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APOBEC3G (A3G), a member of cytidine deaminase family, has potent anti-human immunodeficiency virus type 1 (HIV-1) activity. It has been demonstrated that alpha interferon (IFN-α) can significantly enhance the expression of A3G in human primary resting CD4+ T-cells, macrophages and primary hepatocytes, subsequently decreasing their viral susceptibility. Plasmacytoid dendritic cells (pDCs) are key effectors in innate host immunity, mediating adaptive immune responses and stimulating IFN-α production in reaction to various stimuli. In this report, we demonstrate that IFN-α, either exogenously added to- or endogenously secreted by pDCs, can enhance the expression of A3G and its family members such as A3A, A3C and A3F. We have also shown that IFN-α can inhibit HIV-1 expression in pDCs. This inhibitory effect could be countered by addition of an A3G-specific short interfering RNA, indicating that IFN-α-induced A3G plays a key role in mediating pDCs response to HIV-1. Given the central role played by pDCs in orchestrating the IFN-α/A3G intercellular network and intracellular signal pathway, our data indicate that pDCs themselves are also protected by an IFN-α/A3G-mediated innate immunity barrier from HIV-1 infection.
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Krisko, John F., Nurjahan Begum, Caroline E. Baker, John L. Foster, and J. Victor Garcia. "APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication." Journal of Virology 90, no. 9 (February 24, 2016): 4681–95. http://dx.doi.org/10.1128/jvi.03275-15.
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ABSTRACTThe multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF(JRCSF) expressing avifgene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing avifgene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF withvifmutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforcedvifmutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations invifbut instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations inpolviral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA.IMPORTANCEVif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly, fixation of mutations that replaced H42/43D or W79S in viral RNA lagged behind the appearance of high viral loads. In one exceptional JRCSFvifH42/43D infection,vifwas unchanged but replication proceeded after a long delay. These results suggest that Vif binds and inhibits the non-cytosine deaminase activities of intact A3G and intact A3F, allowing JRCSFvifH42/43D and JRCSFvifW79S to replicate with reduced fitness. Subsequently, enhanced Vif function is acquired by enforced mutations. In infected cells, JRCSFΔvif and JRCSFvifH42/43DW79S are exposed to active A3F and A3G and fail to replicate. JRCSFvifH42/43D Vif degrades A3F and, in some cases, overcomes A3G mutagenic activity to replicate. Vif may have evolved to inhibit A3F and A3G by stoichiometric binding and subsequently acquired the ability to target these proteins to proteasomes.
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Koning, Fransje A., Edmund N. C. Newman, Eun-Young Kim, Kevin J. Kunstman, Steven M. Wolinsky, and Michael H. Malim. "Defining APOBEC3 Expression Patterns in Human Tissues and Hematopoietic Cell Subsets." Journal of Virology 83, no. 18 (July 8, 2009): 9474–85. http://dx.doi.org/10.1128/jvi.01089-09.
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ABSTRACT Human APOBEC3 enzymes are cellular DNA cytidine deaminases that inhibit and/or mutate a variety of retroviruses, retrotransposons, and DNA viruses. Here, we report a detailed examination of human APOBEC3 gene expression, focusing on APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) infection but are suppressed by HIV-1 Vif. A3G and A3F are expressed widely in hematopoietic cell populations, including T cells, B cells, and myeloid cells, as well as in tissues where mRNA levels broadly correlate with the lymphoid cell content (gonadal tissues are exceptions). By measuring mRNA copy numbers, we find that A3G mRNA is ∼10-fold more abundant than A3F mRNA, implying that A3G is the more significant anti-HIV-1 factor in vivo. A3G and A3F levels also vary between donors, and these differences are sustained over 12 months. Responses to T-cell activation or cytokines reveal that A3G and A3F mRNA levels are induced ∼10-fold in macrophages and dendritic cells (DCs) by alpha interferon (IFN-α) and ∼4-fold in naïve CD4+ T cells. However, immunoblotting revealed that A3G protein levels are induced by IFN-α in macrophages and DCs but not in T cells. In contrast, T-cell activation and IFN-γ had a minimal impact on A3G or A3F expression. Finally, we noted that A3A mRNA expression and protein expression are exquisitely sensitive to IFN-α induction in CD4+ T cells, macrophages, and DCs but not to T-cell activation or other cytokines. Given that A3A does not affect HIV-1 infection, these observations imply that this protein may participate in early antiviral innate immune responses.
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Perdiguero, Beatriz, María M. Lorenzo, and Rafael Blasco. "Vaccinia Virus A34 Glycoprotein Determines the Protein Composition of the Extracellular Virus Envelope." Journal of Virology 82, no. 5 (December 19, 2007): 2150–60. http://dx.doi.org/10.1128/jvi.01969-07.
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ABSTRACT The outer envelope of the extracellular form of vaccinia virus contains five virus-encoded proteins, F13, A33, A34, A56, and B5, that, with the exception of A56, are implicated in virus egress or infectivity. A34, a type II transmembrane glycoprotein, is involved in the induction of actin tails, the release of enveloped virus from the surfaces of infected cells, and the disruption of the virus envelope after ligand binding prior to virus entry. To investigate interactions between A34 and other envelope proteins, a recombinant vaccinia virus (vA34RHA) expressing an epitope-tagged version of A34 (A34HA) was constructed by appending an epitope from influenza virus hemagglutinin to the C terminus of A34. Complexes of A34HA with B5 and A36, but not with A33 or F13, were detected in vA34RHA-infected cells. A series of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A34. Both the cytoplasmic and the transmembrane domains of B5 were dispensable for binding to A34. Most of the extracellular domain of B5, which contains four short consensus repeats homologous to complement control proteins, was sufficient for A34 interaction, indicating that both proteins interact through their ectodomains. Immunofluorescence experiments on cells infected with A34-deficient virus indicated that A34 is required for efficient targeting of B5, A36, and A33 into wrapped virions. Consistent with this observation, the envelope of A34-deficient virus contained normal amounts of F13 but decreased amounts of A33 and B5 with respect to the parental WR virus. These results point to A34 as a major determinant in the protein composition of the vaccinia virus envelope.
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Stupfler, Benjamin, Cédric Verriez, Sarah Gallois-Montbrun, Roland Marquet, and Jean-Christophe Paillart. "Degradation-Independent Inhibition of APOBEC3G by the HIV-1 Vif Protein." Viruses 13, no. 4 (April 3, 2021): 617. http://dx.doi.org/10.3390/v13040617.
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The ubiquitin–proteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.
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Talluri, Srikanth, Mehmet Kemal Samur, Jialan Shi, Rao Prabhala, Hervé Avet-Loiseau, Masood A. Shammas, and Nikhil Munshi. "Critical Role for Apobec and Its Interacting Partners in Mediating Mutations and Cell Growth in Multiple Myeloma (MM)." Blood 132, Supplement 1 (November 29, 2018): 4462. http://dx.doi.org/10.1182/blood-2018-99-118441.
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Abstract The APOBEC family of cytidine deaminases include AID (activity induced deaminase) and 10 related APOBEC enzymes (A1,A2,A3A,A3B,A3C,A3D,A3F,A3G,A3H and A4). AID is well studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes. APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Recently, a causal role for the AID/APOBECs in inducing somatic mutations in myeloma has been proposed and we have previously published that APOBEC signature mutations as a frequent event in Myeloma. We have also observed that APOBEC-mediated mutations may account for mutations associated with progression of smoldering myeloma to MM. We further investigated the role of APOBEC in genomic changes in MM and observed that APOBEC expression and activity is elevated in myeloma cell lines as well as patient samples. Using knockdown and over expression approaches, we showed that depletion of APOBECs in myeloma cell lines reduces genomic instability. Following APOBEC3G knock down we observed decreased DNA damage (by g-H2AX), decrease in acquisition of new copy number events over time, and reduced mutational load, strongly suggesting that inhibiting APOBECs could be a potential approach to reduce genome evolution in MM. We next investigated the effect of APOBEC inhibition on myeloma cell proliferation. We observed that Sh-RNA-based APOBEC knock down in MM1S and H929 MM cell lines, led to significant inhibition of MM cell proliferation, and induction of apoptotic cell death. Associated with APOBEC knockdown, we also observed increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1) at both RNA and protein level. By immunoprecipitation we found that APOBEC3G interacts and inhibits the RNA binding protein DEAD-END 1 (DND1), thereby preventing it from inhibiting miR-221-mediated targeting of p27 transcripts. Knockdown of DND1, or over-expression of miR-221 in APOBEC-depleted cells rescued the cell proliferation defects with concomitant decrease in p27 levels. These results show that APOBCs bind to and sequester DND1, leading to miR-221-mediated depletion of p27. In the absence of APOBEC, DND1 prevents the degradation of p27 mRNA, leading to elevated p27 levels and inhibition of cell cycle, suggesting a role for APOBECs in regulating MM cell proliferation that might be independent of its RNA/DNA mutator function. Taken together, these results indicate a significant functional role for APOBECs both in genome evolution as well as cell growth in myeloma and may constitute an important therapeutic target. Disclosures Munshi: OncoPep: Other: Board of director.
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Perdiguero, Beatriz, and Rafael Blasco. "Interaction between Vaccinia Virus Extracellular Virus Envelope A33 and B5 Glycoproteins." Journal of Virology 80, no. 17 (September 1, 2006): 8763–77. http://dx.doi.org/10.1128/jvi.00598-06.
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ABSTRACT The extracellular form of vaccinia virus acquires its outer envelope by wrapping with cytoplasmic membranes that contain at least seven virus-encoded proteins, of which four are glycoproteins. We searched for interactions between the vaccinia virus A33 glycoprotein and proteins A34, A36, B5, F12, and F13. First, when myc epitope-tagged A33 was expressed in combination with other envelope proteins, A33 colocalized with B5 and A36, suggesting that direct A33-B5 and A33-A36 interactions occur in the absence of infection. A recombinant vaccinia virus (vA33Rmyc) was constructed by introduction of the myc-tagged A33 version (A33myc) into A33-deficient vaccinia virus. A33myc partially restored plaque formation and colocalized with enveloped virions in infected cells. Coimmunoprecipitation experiments with extracts of vA33Rmyc-infected cells confirmed the existence of a physical association of A33 with A36 and B5. Of these, the A33-B5 interaction is a novel finding, whereas the interaction between A33 and A36 has been previously characterized. A collection of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A33. Both the cytoplasmic domain and most of the extracellular domain, but not the transmembrane domain, of the B5 protein were dispensable for binding to A33. Mutations in the extracellular portions of B5 and A33 that enhance extracellular virus release did not affect the interaction between the two. In contrast, substituting the B5 transmembrane domain with that of the vesicular stomatitis virus G glycoprotein prevented the association with A33. Immunofluorescence experiments on virus mutants indicated that B5 is required for efficient targeting of A33 into enveloped virions. These results point to the transmembrane domain of B5 as the major determinant of the A33-B5 interaction and demonstrate that protein-protein interactions are crucial in determining the composition of the virus envelope.
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Barzak, Fareeda M., Timothy M. Ryan, Maksim V. Kvach, Harikrishnan M. Kurup, Hideki Aihara, Reuben S. Harris, Vyacheslav V. Filichev, Elena Harjes, and Geoffrey B. Jameson. "Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor." Viruses 13, no. 2 (February 12, 2021): 290. http://dx.doi.org/10.3390/v13020290.
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In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.
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Jost, Stéphanie, Priscilla Turelli, Bastien Mangeat, Ulrike Protzer, and Didier Trono. "Induction of Antiviral Cytidine Deaminases Does Not Explain the Inhibition of Hepatitis B Virus Replication by Interferons." Journal of Virology 81, no. 19 (July 25, 2007): 10588–96. http://dx.doi.org/10.1128/jvi.02489-06.
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ABSTRACT Interferons (IFNs) play a major role in the control of hepatitis B virus (HBV), whether as endogenous cytokines limiting the spread of the virus during the acute phase of the infection or as drugs for the treatment of its chronic phase. However, the mechanism by which IFNs inhibit HBV replication has so far remained elusive. Here, we show that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G (A3G) and, to a lesser extent, that of APOBEC3F (A3F) and APOBEC3B (A3B) but not that of two other cytidine deaminases also endowed with anti-HBV activity, activation-induced cytidine deaminase (AID), and APOBEC1. Most importantly, we reveal that blocking A3B, A3F, and A3G by combining RNA interference and the virion infectivity factor (Vif) protein of human immunodeficiency virus does not abrogate the inhibitory effect of IFNs on HBV. We conclude that these cytidine deaminases are not essential effectors of IFN in its action against this pathogen.
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Zhen, Anjie, Tao Wang, Ke Zhao, Yong Xiong, and Xiao-Fang Yu. "A Single Amino Acid Difference in Human APOBEC3H Variants Determines HIV-1 Vif Sensitivity." Journal of Virology 84, no. 4 (November 25, 2009): 1902–11. http://dx.doi.org/10.1128/jvi.01509-09.
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ABSTRACT Several variants of APOBEC3H (A3H) have been identified in different human populations. Certain variants of this protein are particularly potent inhibitors of retrotransposons and retroviruses, including HIV-1. However, it is not clear whether HIV-1 Vif can recognize and suppress the antiviral activity of A3H variants, as it does with other APOBEC3 proteins. We now report that A3H_Haplotype II (HapII), a potent inhibitor of HIV-1 in the absence of Vif, can indeed be degraded by HIV-1 Vif. Vif-induced degradation of A3H_HapII was blocked by the proteasome inhibitor MG132 and a Cullin5 (Cul5) dominant negative mutant. In addition, Vif mutants that were incapable of assembly with the host E3 ligase complex factors Cul5, ElonginB, and ElonginC were also defective for A3H_HapII suppression. Although we found that Vif hijacks the same E3 ligase to degrade A3H_HapII as it does to inactivate APOBEC3G (A3G) and APOBEC3F (A3F), more Vif motifs were involved in A3H_HapII inactivation than in either A3G or A3F suppression. In contrast to A3H_HapII, A3H_Haplotype I (HapI), which differs in only three amino acids from A3H_HapII, was resistant to HIV-1 Vif-mediated degradation. We also found that residue 121 was critical for determining A3H sensitivity and binding to HIV-1 Vif.
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Luo, Kun, Tao Wang, Bindong Liu, Chunjuan Tian, Zuoxiang Xiao, John Kappes, and Xiao-Fang Yu. "Cytidine Deaminases APOBEC3G and APOBEC3F Interact with Human Immunodeficiency Virus Type 1 Integrase and Inhibit Proviral DNA Formation." Journal of Virology 81, no. 13 (April 11, 2007): 7238–48. http://dx.doi.org/10.1128/jvi.02584-06.
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ABSTRACT APOBEC3G (A3G) is a single-stranded DNA cytidine deaminase that targets retroviral minus-strand DNA and has potent antiviral activity against diverse retroviruses. However, the mechanisms of A3G antiviral functions are incompletely understood. Here we demonstrate that A3G, A3F, and, to a lesser extent, the noncatalytic A3GC291S block human immunodeficiency virus type 1 (HIV-1) replication by interfering with proviral DNA formation. In HIV-1 virions, A3G interacted with HIV-1 integrase and nucleocapsid, key viral factors for reverse transcription and integration. Unlike A3G, the weak antiviral A3C cytidine deaminase did not interact with either of these factors and did not affect viral reverse transcription or proviral DNA formation. Thus, multiple steps of the HIV-1 replication cycle, most noticeably the formation of proviral DNA, are inhibited by both cytidine deamination-dependent and -independent mechanisms.
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Delviks-Frankenberry, Krista A., Belete A. Desimmie, and Vinay K. Pathak. "Structural Insights into APOBEC3-Mediated Lentiviral Restriction." Viruses 12, no. 6 (May 27, 2020): 587. http://dx.doi.org/10.3390/v12060587.
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Mammals have developed clever adaptive and innate immune defense mechanisms to protect against invading bacterial and viral pathogens. Human innate immunity is continuously evolving to expand the repertoire of restriction factors and one such family of intrinsic restriction factors is the APOBEC3 (A3) family of cytidine deaminases. The coordinated expression of seven members of the A3 family of cytidine deaminases provides intrinsic immunity against numerous foreign infectious agents and protects the host from exogenous retroviruses and endogenous retroelements. Four members of the A3 proteins—A3G, A3F, A3H, and A3D—restrict HIV-1 in the absence of virion infectivity factor (Vif); their incorporation into progeny virions is a prerequisite for cytidine deaminase-dependent and -independent activities that inhibit viral replication in the host target cell. HIV-1 encodes Vif, an accessory protein that antagonizes A3 proteins by targeting them for polyubiquitination and subsequent proteasomal degradation in the virus producing cells. In this review, we summarize our current understanding of the role of human A3 proteins as barriers against HIV-1 infection, how Vif overcomes their antiviral activity, and highlight recent structural and functional insights into A3-mediated restriction of lentiviruses.
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Chen, Gongying, Zhiwen He, Tao Wang, Rongzhen Xu, and Xiao-Fang Yu. "A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G." Journal of Virology 83, no. 17 (June 17, 2009): 8674–82. http://dx.doi.org/10.1128/jvi.00653-09.
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ABSTRACT The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K22x3K26x3Y30x9YRHHY44) and a cluster of hydrophobic residues (V55xIPLx4-5LxΦx2YWxL72) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.
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Dang, Ying, Xiaojun Wang, Tao Zhou, Ian A. York, and Yong-Hui Zheng. "Identification of a Novel WxSLVK Motif in the N Terminus of Human Immunodeficiency Virus and Simian Immunodeficiency Virus Vif That Is Critical for APOBEC3G and APOBEC3F Neutralization." Journal of Virology 83, no. 17 (June 17, 2009): 8544–52. http://dx.doi.org/10.1128/jvi.00651-09.
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ABSTRACT The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A 14DRMR17 domain binds to A3F, 40YRHHY44 binds to A3G, and 69YxxL72 binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved 21WxSLVK26 (x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, 21WxSLVK26 is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.
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Breiman, Adrien, David C. J. Carpentier, Helen A. Ewles, and Geoffrey L. Smith. "Transport and stability of the vaccinia virus A34 protein is affected by the A33 protein." Journal of General Virology 94, no. 4 (April 1, 2013): 720–25. http://dx.doi.org/10.1099/vir.0.049486-0.
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Vaccinia virus (VACV) has two infectious forms called intracellular mature virus and extracellular enveloped virus (EEV). Two of the seven viral proteins in the EEV outer envelope, A33 and A34, are type II membrane glycoproteins that each interact with another EEV protein called B5; however, evidence for direct A33–A34 interaction is lacking. The localization and stability of A34 is affected by B5 and here data are presented showing that A34 is also affected by A33. In the absence of A33, just as without B5, the level, localization and glycosylation profile of A34 was altered. However, the glycosylation profile of A34 without A33 is different to that observed in the absence of B5, and A34 accumulates in the Golgi apparatus rather than in the endoplasmic reticulum. Thus, A34 requires more than one other EEV protein for its processing and cellular transport.
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Russell, Rebecca A., and Vinay K. Pathak. "Identification of Two Distinct Human Immunodeficiency Virus Type 1 Vif Determinants Critical for Interactions with Human APOBEC3G and APOBEC3F." Journal of Virology 81, no. 15 (May 23, 2007): 8201–10. http://dx.doi.org/10.1128/jvi.00395-07.
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ABSTRACT Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development because inhibiting the interactions could allow the host defense mechanism to control HIV-1 replication. It was recently reported that the Vif amino acids D14RMR17 are important for functional interaction and degradation of the previously identified Vif-resistant mutant of A3G (D128K-A3G). However, the Vif determinants important for functional interaction with A3G and A3F have not been fully characterized. To identify these determinants, we performed an extensive mutational analysis of HIV-1 Vif. Our analysis revealed two distinct Vif determinants, amino acids Y40RHHY44 and D14RMR17, which are essential for binding to A3G and A3F, respectively. Interestingly, mutation of the A3G-binding region increased Vif's ability to suppress A3F. Vif binding to D128K-A3G was also dependent on the Y40RHHY44 region but not the D14RMR17 region. Consistent with previous observations, subsequent neutralization of the D128K-A3G antiviral activity required substitution of Vif determinant D14RMR17 with SEMQ, similar to the SERQ amino acids in simian immunodeficiency virus SIVAGM Vif, which is capable of neutralizing D128K-A3G. These studies are the first to clearly identify two distinct regions of Vif that are critical for independent interactions with A3G and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins.
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Miyagi, Eri, Charles R. Brown, Sandrine Opi, Mohammad Khan, Ritu Goila-Gaur, Sandra Kao, Robert C. Walker, Vanessa Hirsch, and Klaus Strebel. "Stably Expressed APOBEC3F Has Negligible Antiviral Activity." Journal of Virology 84, no. 21 (August 11, 2010): 11067–75. http://dx.doi.org/10.1128/jvi.01249-10.
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ABSTRACT APOBEC3F (A3F) is a member of the family of cytidine deaminases that is often coexpressed with APOBEC3G (A3G) in cells susceptible to HIV infection. A3F has been shown to have strong antiviral activity in transient-expression studies, and together with A3G, it is considered the most potent cytidine deaminase targeting HIV. Previous analyses suggested that the antiviral properties of A3F can be dissociated from its catalytic deaminase activity. We were able to confirm the deaminase-independent antiviral activity of exogenously expressed A3F; however, we also noted that exogenous expression was associated with very high A3F mRNA and protein levels. In analogy to our previous study of A3G, we produced stable HeLa cell lines constitutively expressing wild-type or deaminase-defective A3F at levels that were more in line with the levels of endogenous A3F in H9 cells. A3F expressed in stable HeLa cells was packaged into Vif-deficient viral particles with an efficiency similar to that of A3G and was properly targeted to the viral nucleoprotein complex. Surprisingly, however, neither wild-type nor deaminase-defective A3F inhibited HIV-1 infectivity. These results imply that the antiviral activity of endogenous A3F is negligible compared to that of A3G.
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Russell, Rebecca A., Jessica Smith, Rebekah Barr, Darshana Bhattacharyya, and Vinay K. Pathak. "Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif." Journal of Virology 83, no. 4 (November 26, 2008): 1992–2003. http://dx.doi.org/10.1128/jvi.01621-08.
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ABSTRACT Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their degradation. Thus, the Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development, as inhibiting these interactions could allow the host defense mechanism to control HIV-1 replication. Recently, it has been reported that amino acids 105 to 156 of A3G are involved in the interaction with Vif; however, to date, the region of A3F involved in Vif binding has not been identified. Using our previously reported Vif mutants that are capable of binding to only A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that the A3G domain that interacts with the Vif YRHHY region is located between amino acids 126 and 132 of A3G, which is consistent with the conclusions reported in previous studies. The A3F domain that interacts with the Vif DRMR region did not occur in the homologous domain but instead was located between amino acids 283 and 300 of A3F. These studies are the first to identify the A3F domain that interacts with the Vif DRMR region and show that distinct domains of A3G and A3F interact with different Vif regions. Pharmacological inhibition of either or both of these Vif-A3 interactions should prevent the degradation of the APOBEC3 proteins and could be used as a therapy against HIV-1.
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Li, Zusheng, Anthony J. Clarke, and Terry J. Beveridge. "Gram-Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5478–83. http://dx.doi.org/10.1128/jb.180.20.5478-5483.1998.
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ABSTRACT Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter,Enterobacter, Escherichia,Klebsiella, Morganella, Proteus,Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected. Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity.
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Mbisa, Jean L., Wei Bu, and Vinay K. Pathak. "APOBEC3F and APOBEC3G Inhibit HIV-1 DNA Integration by Different Mechanisms." Journal of Virology 84, no. 10 (March 10, 2010): 5250–59. http://dx.doi.org/10.1128/jvi.02358-09.
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ABSTRACT APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3′ long terminal repeat (3′-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3′ processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3′-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.
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Gallois-Montbrun, Sarah, Rebecca K. Holmes, Chad M. Swanson, Mireia Fernández-Ocaña, Helen L. Byers, Malcolm A. Ward, and Michael H. Malim. "Comparison of Cellular Ribonucleoprotein Complexes Associated with the APOBEC3F and APOBEC3G Antiviral Proteins." Journal of Virology 82, no. 11 (March 26, 2008): 5636–42. http://dx.doi.org/10.1128/jvi.00287-08.
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ABSTRACT The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F (APOBEC3F [A3F]) and A3G proteins are effective inhibitors of infection by various retroelements and share ∼50% amino acid sequence identity. We therefore undertook comparative analyses of the protein and RNA compositions of A3F- and A3G-associated ribonucleoprotein complexes (RNPs). Like A3G, A3F is found associated with a complex array of cytoplasmic RNPs and can accumulate in RNA-rich cytoplasmic microdomains known as mRNA processing bodies or stress granules. While A3F RNPs display greater resistance to disruption by RNase digestion, the major protein difference is the absence of the Ro60 and La autoantigens. Consistent with this, A3F RNPs also lack a number of small polymerase III RNAs, including the RoRNP-associated Y RNAs, as well as 7SL RNA. Alu RNA is, however, present in A3F and A3G RNPs, and both proteins suppress Alu element retrotransposition. Thus, we define a number of subtle differences between the RNPs associated with A3F and A3G and speculate that these contribute to functional differences that have been described for these proteins.
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Albin, John S., Guylaine Haché, Judd F. Hultquist, William L. Brown, and Reuben S. Harris. "Long-Term Restriction by APOBEC3F Selects Human Immunodeficiency Virus Type 1 Variants with Restored Vif Function." Journal of Virology 84, no. 19 (August 4, 2010): 10209–19. http://dx.doi.org/10.1128/jvi.00632-10.
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ABSTRACT Tandem stop mutations K26X and H27X in human immunodeficiency virus type 1 (HIV-1) vif compromise virus replication in human T-cell lines that stably express APOBEC3F (A3F) or APOBEC3G (A3G). We previously reported that partial resistance to A3G could develop in these Vif-deficient viruses through a nucleotide A200-to-T/C transversion and a vpr null mutation, but these isolates were still susceptible to restriction by A3F. Here, long-term selection experiments were done to determine how these A3G-selected isolates might evolve to spread in the presence of A3F. We found that A3F, like A3G, is capable of potent, long-term restriction that eventually selects for heritable resistance. In all 7 instances, the selected isolates had restored Vif function to cope with A3F activity. In two isolates, Vif Q26-Q27 and Y26-Q27, the resistance phenotype recapitulated in molecular clones, but when the selected vif alleles were analyzed in the context of an otherwise wild-type viral background, a different outcome emerged. Although HIV-1 clones with Vif Q26-Q27 or Y26-Q27 were fully capable of overcoming A3F, they were now susceptible to restriction by A3G. Concordant with prior studies, a lysine at position 26 proved essential for A3G neutralization. These data combine to indicate that A3F and A3G exert at least partly distinct selective pressures and that Vif function may be essential for the virus to replicate in the presence of A3F.
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Paprotka, Tobias, Narasimhan J. Venkatachari, Chawaree Chaipan, Ryan Burdick, Krista A. Delviks-Frankenberry, Wei-Shau Hu, and Vinay K. Pathak. "Inhibition of Xenotropic Murine Leukemia Virus-Related Virus by APOBEC3 Proteins and Antiviral Drugs." Journal of Virology 84, no. 11 (March 24, 2010): 5719–29. http://dx.doi.org/10.1128/jvi.00134-10.
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ABSTRACT Xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, has been isolated from human prostate cancer tissue and from activated CD4+ T cells and B cells of patients with chronic fatigue syndrome, suggesting an association between XMRV infection and these two diseases. Since APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of murine leukemia virus and Vif-deficient human immunodeficiency virus type 1 (HIV-1), are expressed in human CD4+ T cells and B cells, we sought to determine how XMRV evades suppression of replication by APOBEC3 proteins. We found that expression of A3G, A3F, or murine A3 in virus-producing cells resulted in their virion incorporation, inhibition of XMRV replication, and G-to-A hypermutation of the viral DNA with all three APOBEC3 proteins. Quantitation of A3G and A3F mRNAs indicated that, compared to the human T-cell lines CEM and H9, prostate cell lines LNCaP and DU145 exhibited 50% lower A3F mRNA levels, whereas A3G expression in 22Rv1, LNCaP, and DU145 cells was nearly undetectable. XMRV proviral genomes in LNCaP and DU145 cells were hypermutated at low frequency with mutation patterns consistent with A3F activity. XMRV proviral genomes were extensively hypermutated upon replication in A3G/A3F-positive T cells (CEM and H9), but not in A3G/A3F-negative cells (CEM-SS). We also observed that XMRV replication was susceptible to the nucleoside reverse transcriptase (RT) inhibitors zidovudine (AZT) and tenofovir and the integrase inhibitor raltegravir. In summary, the establishment of XMRV infection in patients may be dependent on infection of A3G/A3F-deficient cells, and cells expressing low levels of A3G/A3F, such as prostate cancer cells, may be ideal producers of infectious XMRV. Furthermore, the anti-HIV-1 drugs AZT, tenofovir, and raltegravir may be useful for treatment of XMRV infection.
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Dang, Ying, Roderick W. Davis, Ian A. York, and Yong-Hui Zheng. "Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity." Journal of Virology 84, no. 11 (March 24, 2010): 5741–50. http://dx.doi.org/10.1128/jvi.00079-10.
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ABSTRACT The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, 81LGxGxSIEW89 and 171EDRWN175. Within the 81LGxGxSIEW89 sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues 86SIEW89 affect Vif binding to A3F, A3G, and Cul5. Accordingly, this 81LGxGxSIEW89 sequence was designated the 81LGxGxxIxW89 domain. Within the 171EDRWN175 sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated 171EDRW174. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, 81LGxGxxIxW89 and 171EDRW174 are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.
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Tian, Chunjuan, Xianghui Yu, Wei Zhang, Tao Wang, Rongzhen Xu, and Xiao-Fang Yu. "Differential Requirement for Conserved Tryptophans in Human Immunodeficiency Virus Type 1 Vif for the Selective Suppression of APOBEC3G and APOBEC3F." Journal of Virology 80, no. 6 (March 15, 2006): 3112–15. http://dx.doi.org/10.1128/jvi.80.6.3112-3115.2006.
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ABSTRACT APOBEC3G (A3G) and related cytidine deaminases, such as APOBEC3F (A3F), are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus type 1 (HIV-1) requires suppression of multiple cytidine deaminases by Vif. Whether HIV-1 Vif recognizes various APOBEC3 proteins through a common mechanism is unclear. The domains in Vif that mediate APOBEC3 recognitions are also poorly defined. The N-terminal region of HIV-1 Vif is unusually rich in Trp residues, which are highly conserved. In the present study, we examined the role of these Trp residues in the suppression of APOBEC3 proteins by HIV-1 Vif. We found that most of the highly conserved Trp residues were required for efficient suppression of both A3G and A3F, but some of these residues were selectively required for the suppression of A3F but not A3G. Mutant Vif molecules in which Ala was substituted for Trp79 and, to a lesser extent, for Trp11 remained competent for A3G interaction and its suppression; however, they were defective for A3F interaction and therefore could not efficiently suppress the antiviral activity of A3F. Interestingly, while the HIV-1 Vif-mediated degradation of A3G was not affected by the different C-terminal tag peptides, that of A3F was significantly influenced by its C-terminal tags. These data indicate that the mechanisms by which HIV-1 Vif recognizes its target molecules, A3G and A3F, are not identical. The fact that several highly conserved residues in Vif are required for the suppression of A3F but not that of A3G suggests a critical role for A3F in the restriction of HIV-1 in vivo.
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Liu, Bindong, Phuong Thi Nguyen Sarkis, Kun Luo, Yunkai Yu, and Xiao-Fang Yu. "Regulation of Apobec3F and Human Immunodeficiency Virus Type 1 Vif by Vif-Cul5-ElonB/C E3 Ubiquitin Ligase." Journal of Virology 79, no. 15 (August 1, 2005): 9579–87. http://dx.doi.org/10.1128/jvi.79.15.9579-9587.2005.
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ABSTRACT The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.
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Nakashima, Masaaki, Hirotaka Ode, Takashi Kawamura, Shingo Kitamura, Yuriko Naganawa, Hiroaki Awazu, Shinya Tsuzuki, et al. "Structural Insights into HIV-1 Vif-APOBEC3F Interaction." Journal of Virology 90, no. 2 (November 4, 2015): 1034–47. http://dx.doi.org/10.1128/jvi.02369-15.
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ABSTRACTThe HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.IMPORTANCEHIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.
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Mulder, Lubbertus C. F., Marcel Ooms, Susan Majdak, Jordan Smedresman, Caitlin Linscheid, Ariana Harari, Andrea Kunz, and Viviana Simon. "Moderate Influence of Human APOBEC3F on HIV-1 Replication in Primary Lymphocytes." Journal of Virology 84, no. 18 (June 30, 2010): 9613–17. http://dx.doi.org/10.1128/jvi.02630-09.
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ABSTRACT Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.
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Doceul, Virginie, Michael Hollinshead, Adrien Breiman, Kathlyn Laval, and Geoffrey L. Smith. "Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions." Journal of General Virology 93, no. 9 (September 1, 2012): 1876–86. http://dx.doi.org/10.1099/vir.0.043943-0.
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Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.
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Dang, Ying, Xiaojun Wang, Ian A. York, and Yong-Hui Zheng. "Identification of a Critical T(Q/D/E)x5ADx2(I/L) Motif from Primate Lentivirus Vif Proteins That Regulate APOBEC3G and APOBEC3F Neutralizing Activity." Journal of Virology 84, no. 17 (June 30, 2010): 8561–70. http://dx.doi.org/10.1128/jvi.00960-10.
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ABSTRACT Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x5ADx2(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif's neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x5ADx2(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.
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Bogerd, Hal P., Anand V. R. Kornepati, Joy B. Marshall, Edward M. Kennedy, and Bryan R. Cullen. "Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): E7249—E7256. http://dx.doi.org/10.1073/pnas.1516305112.
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Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such “synergistic activation mediators” can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.
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De la O Olán, Micaela, Eduardo Espitia Rangel, Héctor Eduardo Villaseñor Mir, José Domingo Molina Galán, Higinio López Sánchez, Amalio Santacruz Varela, and Roberto Javier Peña Bautista. "Proteínas del gluten y reología de trigos harineros mexicanos influeciados por factores ambientales y genotípicos." Pesquisa Agropecuária Brasileira 45, no. 9 (September 2010): 989–96. http://dx.doi.org/10.1590/s0100-204x2010000900008.
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El objetivo de este estudio fue conocer el efecto de factores ambientales y genotípicos sobre los parámetros de calidad industrial y sobre la cantidad y relación de proteínas monoméricas y poliméricas del gluten en 24 líneas recombinantes de trigos harineros de temporal. El cultivo se desarrolló en cinco condiciones ambientales generadas por manejo agronómico, ciclo otoño-invierno 2006/2007, en Roque, Guanajuato, México. Se evaluaron el tiempo de amasado (TMA), fuerza (ALVW), extensibilidad (ALVPL) de la masa, fracción rica en gliadina (50PS) y en glutenina (50PI), y su relación (50PS/50PI). Las mejores combinaciones de gluteninas de alto y bajo peso molecular para TMA y ALVW fueron los genotipos con 1, 17+18, 5+10/Glu-A3c, Glu-B3g, Glu-D3b; 1, 17+18, 5+10/Glu-A3c, Glu-B3h, Glu-D3b, y 2*, 17+18, 5+10/Glu-A3c, Glu-B3g, Glu-D3b; para ALVPL, 2*, 17+18, 2+12/Glu-A3e, Glu-B3h, Glu-D3b; para 50PS, 2*, 17+18, 2+12/Glu-A3e, Glu-B3h, Glu-D3b; y 1, 17+18, 5+10/Glu-A3e, Glu-B3h, Glu-D3b. La relación 50PS/50PI fue mayor en genotipos con 2*, 17+18, 2+12/Glu-A3e, Glu-B3g, Glu-D3b. El TMA es mayor cuando aumenta la temperatura y la mejor ALVPL se obtiene en el ambiente bajo condiciones normales. La fracción 50PS y la relación 50PS/50PI son mayores cuando se realiza la fertilización con azufre, y se obtiene incremento de 50PI con riego limitado y aumento de temperaturas durante el llenado de grano.
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Udquim, Krizia-Ivana, Clara Zettelmeyer, A. Rouf Banday, Seraph Han-Yin Lin, and Ludmila Prokunina-Olsson. "APOBEC3B expression in breast cancer cell lines and tumors depends on the estrogen receptor status." Carcinogenesis 41, no. 8 (January 13, 2020): 1030–37. http://dx.doi.org/10.1093/carcin/bgaa002.
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Abstract Increased exposure to estrogen is associated with an elevated risk of breast cancer. Considering estrogen as a possible mutagen, we hypothesized that exposure to estrogen alone or in combination with the DNA-damaging chemotherapy drug, cisplatin, could induce expression of genes encoding enzymes involved in APOBEC-mediated mutagenesis. To test this hypothesis, we measured the expression of APOBEC3A (A3A) and APOBEC3B (A3B) genes in two breast cancer cell lines treated with estradiol, cisplatin or their combination. These cell lines, T-47D (ER+) and MDA-MB-231 (ER−), differed by the status of the estrogen receptor (ER). Expression of A3A was not detectable in any conditions tested, while A3B expression was induced by treatment with cisplatin and estradiol in ER+ cells but was not affected by estradiol in ER− cells. In The Cancer Genome Atlas, expression of A3B was significantly associated with genotypes of a regulatory germline variant rs17000526 upstream of the APOBEC3 cluster in 116 ER− breast tumors (P = 0.006) but not in 387 ER+ tumors (P = 0.48). In conclusion, we show that in breast cancer cell lines, A3B expression was induced by estradiol in ER+ cells and by cisplatin regardless of ER status. In ER+ breast tumors, the effect of estrogen may be masking the association of rs17000526 with A3B expression, which was apparent in ER− tumors. Our results provide new insights into the differential etiology of ER+ and ER− breast cancer and the possible role of A3B in this process through a mitogenic rather than the mutagenic activity of estrogen.
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Fernández, E., J. Oliveira, and M. R. Sancho. "A murine monoclonal antibody detecting HLA-A30 and HLA-A31." Tissue Antigens 37, no. 1 (January 1991): 42–43. http://dx.doi.org/10.1111/j.1399-0039.1991.tb01843.x.
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Falk, Kirsten, Olaf Rötzschke, Masafumi Takiguchi, Blazenka Grahovac, Volker Gnau, Stefan Stevanović, Günther Jung, and Hans-George Rammensee. "Peptide motifs of HLA-A1,-A11,-A31, and-A33 molecules." Immunogenetics 40, no. 3 (July 1994): 238–41. http://dx.doi.org/10.1007/bf00167086.
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Zhang, Zeli, Qinyong Gu, Ananda Ayyappan Jaguva Vasudevan, Manimehalai Jeyaraj, Stanislaw Schmidt, Jörg Zielonka, Mario Perković, et al. "Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C." Journal of Virology 90, no. 22 (August 31, 2016): 10193–208. http://dx.doi.org/10.1128/jvi.01497-16.
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ABSTRACTLentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.IMPORTANCEThe APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.
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Niewiadomska, Anna Maria, Chunjuan Tian, Lindi Tan, Tao Wang, Phuong Thi Nguyen Sarkis, and Xiao-Fang Yu. "Differential Inhibition of Long Interspersed Element 1 by APOBEC3 Does Not Correlate with High-Molecular-Mass-Complex Formation or P-Body Association." Journal of Virology 81, no. 17 (June 20, 2006): 9577–83. http://dx.doi.org/10.1128/jvi.02800-06.
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ABSTRACT The human cytidine deaminase APOBEC3G (A3G) and other APOBEC3 proteins exhibit differential inhibitory activities against diverse endogenous retroelements and retroviruses, including Vif-deficient human immunodeficiency virus type 1. The potential inhibitory activity of human APOBEC proteins against long interspersed element 1 (LINE-1) has not been fully evaluated. Here, we demonstrate inhibition of LINE-1 by multiple human APOBEC3 cytidine deaminases, including previously unreported activity for A3DE and A3G. More ancient members of APOBEC, cytidine deaminases AID and APOBEC2, had no detectable activity against LINE-1. A3A, which did not form high-molecular-mass (HMM) complexes and interacted poorly with P bodies, was the most potent inhibitor of LINE-1. A3A specifically recognizes LINE-1 RNA but not the other cellular RNAs tested. However, in the presence of LINE-1, A3A became associated with HMM complexes containing LINE-1 RNA. The ability of A3A to recognize LINE-1 RNA required its catalytic domain and was important for its LINE-1 suppression. Although the mechanism of LINE-1 restriction did not seem to involve DNA editing, A3A inhibited the accumulation of nascent LINE-1 DNA, suggesting interference with LINE-1 reverse transcription and/or integration or intracellular movement of LINE-1 ribonucleoprotein. Thus, association with P bodies or cellular HMM complexes could not predict the potency of APOBEC3 anti-LINE-1 activities. The catalytic domain of APOBEC3 proteins may be important for proper folding and target factors such as RNA or protein interaction in addition to cytidine deamination.
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McDaniel, Yumeng Z., Dake Wang, Robin P. Love, Madison B. Adolph, Nazanin Mohammadzadeh, Linda Chelico, and Louis M. Mansky. "Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure." Nucleic Acids Research 48, no. 3 (January 16, 2020): 1353–71. http://dx.doi.org/10.1093/nar/gkz1164.
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Abstract The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5′TC3′ or 5′CT3′ dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.
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Gao, Hui, Rui Li, and Yi Guo. "Arabidopsis aspartic proteases A36 and A39 play roles in plant reproduction." Plant Signaling & Behavior 12, no. 4 (April 3, 2017): e1304343. http://dx.doi.org/10.1080/15592324.2017.1304343.
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Jin, H., J. Yan, R. J. Peña, X. C. Xia, A. Morgounov, L. M. Han, Y. Zhang, and Z. H. He. "Molecular detection of high- and low-molecular-weight glutenin subunit genes in common wheat cultivars from 20 countries using allele-specific markers." Crop and Pasture Science 62, no. 9 (2011): 746. http://dx.doi.org/10.1071/cp11134.
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The composition and quantity of high- and low-molecular-weight glutenin subunits (HMW-GS and LMW-GS) plays an important role in determining the end-use quality of wheat products. In the present study, 718 wheat cultivars and advanced lines from 20 countries were characterised for the HMW-GS and LMW-GS with allele-specific molecular markers. For the Glu-A1 locus, 311 cultivars (43.3%) had the subunit Ax2*, which predominated in cultivars from Canada (83.3%), Romania (91.7%), Russia (72.2%) and USA (72.2%). At Glu-B1 locus, 197 cultivars (27.4%) contained the By8 subunit and its frequency was higher in Japanese (60.0%) and Romanian (62.5%) genotypes than in those from other countries; 264 cultivars (36.8%) carried the By9 subunit, mostly existing in the cultivars from Austria (100.0%), Russia (72.2%), and Serbia (72.7%); the By16 subunit was present in 44 cultivars (6.1%), with a relatively high percentage in Chile (19.5%), whereas almost no cultivars from other countries had this subunit; the frequency of Bx7OE was 3.1%, and was found only in cultivars from Argentina (12.1%), Australia (4.1%), Canada (25.0%), Iran (20.0%), and Japan (30.0%). There were 446 genotypes (62.1%) with the subunit Dx5 at the Glu-D1 locus; high frequencies of Dx5 occurred in cultivars from Hungary (90.0%), Romania (95.8%), and Ukraine (92.3%). At the Glu-A3 locus, the frequencies of Glu-A3a, b, c, d, e, f and g were 2.9, 6.8, 53.2, 12.8, 7.7, 13.8, and 2.4%, respectively. Glu-A3a was detected only in the cultivars from Bulgaria (13.3%), China (12.2%), Germany (2.7%), Iran (6.7%), Mexico (14.3%), Turkey (4.7%), and USA (5.1%); the high frequencies of superior alleles Glu-A3b and d were found in cultivars from Australia (39.7%) and France (24.5%); Glu-A3c was widely distributed in cultivars from all the countries; the high frequencies of Glu-A3e, f and g were detected in cultivars from Argentina (33.3%), Canada (29.2%), and Hungary (20.0%). At the Glu-B3 locus, Glu-B3a, b, c, d, e, f, g, h and i were present in frequencies of 0.4, 22.3, 0.3, 2.8, 1.9, 3.9, 27.2, 18.8, and 7.1%, respectively. Glu-B3a was detected only in cultivars from Argentina (3.0%) and Ukraine (15.4%) cultivars; high frequencies of Glu-B3b and d were found in the cultivars from Romania (62.5%) and Mexico (14.3%); Glu-B3c was detected only in Romanian (8.3%) genotypes; frequencies of e, f, h and i were high in cultivars from Austria (40.0%), China (14.3%), USA (43.0%), and Argentina (33.3%); Glu-B3g was mostly detected in the cultivars from Germany (69.3%), Norway (77.3%), and Serbia (63.6%). The frequency of the 1B·1R translocation was 13.4%; it occurred in cultivars from all the countries except Australia, Austria, Norway, and Serbia. The functional markers applied in this study, in agreement with the results of sodium-dodecylsulfate–polyacrylamide gel electrophoresis, were accurate and stable, and can be used effectively in wheat quality breeding.
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Dawe, Robert D., Lewis N. Mander, John V. Turner, and Pan Xinfu. "Syntheses of c20 gibberellin a36 and a37 methyl esters from gibberellic acid." Tetrahedron Letters 26, no. 46 (January 1985): 5725–28. http://dx.doi.org/10.1016/s0040-4039(01)80931-x.
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Zhang, Wenyan, Juan Du, Kevin Yu, Tao Wang, Xiong Yong, and Xiao-Fang Yu. "Association of Potent Human Antiviral Cytidine Deaminases with 7SL RNA and Viral RNP in HIV-1 Virions." Journal of Virology 84, no. 24 (October 6, 2010): 12903–13. http://dx.doi.org/10.1128/jvi.01632-10.
Full textAbstract:
ABSTRACT 7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{3}^{Lys}\) \end{document} . Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.