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APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis., A. Rouf Banday et al. report targeting alternative splicing of APOBEC3B as a strategy to modulate APOBEC-mediated mutagenesis in cancers. Higher expression of the mutagenic APOBEC3B isoform predicted shorter progression-free survival in bladder cancer patients. Expression of this mutagenic isoform could be decreased by inducing skipping of APOBEC3B exon 5 in cells treated with SF3B1 inhibitor or splice-switching oligos.

Significance For several decades, retroviral core uncoating has been thought to occur in the cytoplasm in coordination with reverse transcription, and while some recent studies have concluded that HIV-1 uncoating occurs at the nuclear envelope during nuclear import, none have concluded that uncoating occurs in the nucleus. Here, we developed methods to study HIV-1 uncoating by direct labeling and quantification of the viral capsid protein associated with infectious viral cores that produced transcriptionally active proviruses. We find that infectious viral cores in the nuclei of infected cells are largely intact and uncoat near their integration sites just before integration. These unexpected findings fundamentally change our understanding of HIV-1 postentry replication events., HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFβ) at 3.9-Å resolution. The structure shows that Vif and CBFβ form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFβ beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of restriction factor cytidine deaminase APOBEC3G (A3G), we developed self-activating lentiviral vectors that efficiently deliver HIV-1 Vif-resistant mutant A3G-D128K to target cells. To circumvent APOBEC3 expression in virus-producing cells, which diminishes virus infectivity, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, retroviral recombination between the direct repeats reconstituted an active A3G-D128K in 89%–98% of transduced cells. Lentiviral vectors that expressed A3G-D128K transduced CD34+ hematopoietic stem and progenitor cells with a high efficiency (>30%). A3G-D128K expression in T cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of several HIV-1 subtypes by C-to-U deamination leading to lethal G-to-A hypermutation and inhibition of reverse transcription. SIVmac239 and HIV-2 were not inhibited, since their Vifs degraded A3G-D128K. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for >3.5 months without detectable resistant virus, suggesting a high genetic barrier for the emergence of A3G-D128K resistance. Because of this, A3G-D128K expression in HIV-1 target cells is a potential anti-HIV gene therapy approach that could be combined with other therapies for the treatment and functional cure of HIV-1 infection., Graphical Abstract

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

Significance Here, we used a fluorescent protein that is free in solution and is trapped in nuclear HIV-1 capsids to demonstrate that the capsids retain integrity and prevent mixing of macromolecules within the viral core and the cellular environment until just before integration. We also found that capsid integrity is maintained until just minutes before disassembly in the nucleus, revealing that uncoating proceeds rapidly after integrity loss. These valuable insights into the early stage of HIV-1 replication indicate that intact HIV-1 capsids are imported through nuclear pores, that reverse transcription is mostly completed within intact capsids, and that preintegration complex-host interactions facilitating integration and target site selection must occur within a short time frame between capsid disassembly and integration., We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to genome packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating genome packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 A and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn2+, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3′ to 5′ directional preference in processive deamination.

Mov10 is a processing body (P-body) protein and an interferon-stimulated gene that can affect replication of retroviruses, hepatitis B virus, and hepatitis C virus (HCV). The mechanism of HCV inhibition by Mov10 is unknown. Here, we investigate the effect of Mov10 on HCV infection and determine the virus life cycle steps affected by changes in Mov10 overexpression. Mov10 overexpression suppresses HCV RNA in both infectious virus and subgenomic replicon systems. Additionally, Mov10 overexpression decreases the infectivity of released virus, unlike control P-body protein DCP1a that has no effect on HCV RNA production or infectivity of progeny virus. Confocal imaging of uninfected cells shows endogenous Mov10 localized at P-bodies. However, in HCV-infected cells, Mov10 localizes in circular structures surrounding cytoplasmic lipid droplets with NS5A and core protein. Mutagenesis experiments show that the RNA binding activity of Mov10 is required for HCV inhibition, while its P-body localization, helicase, and ATP-binding functions are not required. Unexpectedly, endogenous Mov10 promotes HCV replication, as CRISPR-Cas9-based Mov10 depletion decreases HCV replication and infection levels. Our data reveal an important and complex role for Mov10 in HCV replication, which can be perturbed by excess or insufficient Mov10.

The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5′ untranslated region (5′ UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5′ UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in

The MT-4 human T-cell line expresses HTLV-1 Tax and is permissive for replication of an HIV-1 gp41 mutant lacking the cytoplasmic tail. MT-4 cells (lot 150048), distributed by the NIH AIDS Reagent Program (NIH-ARP), were found to be Tax deficient and unable to host replication of the gp41-truncated HIV-1 mutant. These findings, together with short tandem repeat profiling, established that lot 150048 are not bona fide MT-4 cells.

The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.

Genome packaging is an essential step to generate infectious HIV-1 virions and is mediated by interactions between the viral protein Gag and cis-acting elements in the full-length RNA. The sequence necessary and sufficient to allow RNA genome packaging into an HIV-1 particle has not been defined. Here, we used two distinct reporter systems to determine the HIV-1 sequence required for heterologous, non-viral RNAs to be packaged into viral particles. Although the 5’ untranslated region (UTR) of the HIV-1 RNA is known to be important for RNA packaging, we found that its ability to mediate packaging relies heavily on the context of the downstream sequences. Insertion of the 5’ UTR and the first 32-nt of gag into two different reporter RNAs is not sufficient to mediate the packaging of these RNA into HIV-1 particles. However, adding the 5’ half of the gag gene to the 5’ UTR strongly facilitates the packaging of two reporter RNAs; such RNAs can be packaged at >50% of the efficiencies of an HIV-1 near full-length vector. To further examine the role of the gag sequence in RNA packaging, we replaced the 5’ gag sequence in the HIV-1 genome with two codon-optimized gag sequences and found that such substitutions only resulted in a moderate decrease of RNA packaging efficiencies. Taken together, these results indicated that both HIV-1 5’ UTR and the 5’ gag sequence are required for efficient packaging of non-viral RNA into HIV-1 particles, although the gag sequence likely plays an indirect role in genome packaging.

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. IMPORTANCE Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

Retroviruses package two complete RNA genomes into a viral particle but generate only one provirus after each infection. This pseudodiploid replication strategy facilitates frequent recombination, which occurs during DNA synthesis when reverse transcriptase switches templates between two copackaged RNA genomes, generating chimeric DNA. Recombination has played an important role in shaping the current HIV-1 pandemic; however, whether recombination is required for HIV-1 replication is currently unknown. In this report, we examined viral replication when recombination was blocked in defined regions of the HIV-1 genome. We found that blocking recombination reduced viral titers. Furthermore, a significant proportion of the resulting proviruses contained large deletions. Analyses of the deletion junctions indicated that these deletions were the direct consequence of blocking recombination. Thus, our findings illustrate that recombination is a major mechanism to maintain HIV-1 genome integrity. Our study also shows that both obligatory and nonobligatory crossovers occur during reverse transcription, thereby supporting both the forced and dynamic copy-choice models of retroviral recombination. Taken together, our results demonstrate that, in most viruses, both packaged RNA genomes contribute to the genetic information in the DNA form. Furthermore, recombination allows generation of the intact HIV-1 DNA genome and is required for efficient viral replication.

In recent past, it has been seen in many applications that synergism of computational intelligence techniques outperforms over an individual technique. This paper proposes a new hybrid computation model which is a novel synergism of modified evolutionary fuzzy clustering with associated neural networks. It consists of two modules: fuzzy distribution and neural classifier. In first module, mean patterns are distributed into the number of clusters based on the modified evolutionary fuzzy clustering, which leads the basis for network structure selection and learning in associated neural classifier. In second module, training and subsequent generalization is performed by the associated neural networks. The number of associated networks required in the second module will be same as the number of clusters generated in the first module. Whereas, each network contains as many output neurons as the maximum number of members assigned to each cluster. The proposed hybrid model is evaluated over wide spectrum of benchmark problems and real life biometric recognition problems even in presence of real environmental constraints such as noise and occlusion. The results indicate the efficacy of proposed method over related techniques and endeavor promising outcomes for biometric applications with noise and occlusion.

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

The dynamics and regulation of HIV-1 nuclear import and its intranuclear movements after import have not been studied. To elucidate these essential HIV-1 post-entry events, we labeled viral complexes with two fluorescently tagged virion-incorporated proteins (APOBEC3F or integrase), and analyzed the HIV-1 dynamics of nuclear envelope (NE) docking, nuclear import, and intranuclear movements in living cells. We observed that HIV-1 complexes exhibit unusually long NE residence times (1.5±1.6 hrs) compared to most cellular cargos, which are imported into the nuclei within milliseconds. Furthermore, nuclear import requires HIV-1 capsid (CA) and nuclear pore protein Nup358, and results in significant loss of CA, indicating that one of the viral core uncoating steps occurs during nuclear import. Our results showed that the CA-Cyclophilin A interaction regulates the dynamics of nuclear import by delaying the time of NE docking as well as transport through the nuclear pore, but blocking reverse transcription has no effect on the kinetics of nuclear import. We also visualized the translocation of viral complexes docked at the NE into the nucleus and analyzed their nuclear movements and determined that viral complexes exhibited a brief fast phase (, Author summary Although nuclear import of HIV-1 is essential for viral replication, many aspects of this process are currently unknown. Here, we defined the dynamics of HIV-1 nuclear envelope (NE) docking, nuclear import and its relationship to viral core uncoating, and intranuclear movements. We observed that HIV-1 complexes exhibit an unusually long residence time at the NE (∼1.5 hrs) compared to other cellular and viral cargos, and that HIV-1 capsid (CA) and host nuclear pore protein Nup358 are required for NE docking and nuclear import. Soon after import, the viral complexes exhibit a brief fast phase of movement, followed by a long slow phase, during which their movement is similar to that of integrated proviruses, suggesting that they rapidly become tethered to chromatin through interactions that do not require LEDGF/p75. Importantly, we found that NE association and nuclear import is regulated by the CA-cyclophilin A interaction, but not reverse transcription, and that one of the viral core uncoating steps, characterized by substantial loss of CA, occurs concurrently with nuclear import.

Background HIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif–CBFβ interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFβ, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif–CBFβ interactions that are important for Vif function has not been performed. Results Here, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFβ and their ability to induce A3G degradation as well as rescue HIV-1 replication in the presence of A3G. We found that multiple Vif residues are involved in the extensive N-terminal Vif–CBFβ interaction and that the 5WQVMIVW11 region of Vif is the major determinant. A minimum of three alanine substitutions are required to completely abrogate the Vif–CBFβ interaction and Vif’s ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFβ revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction with W5 of Vif to maintain a stable and functional Vif–CBFβ complex. We also determined that CBFβ amino acids 73WQGEQR78, which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif. Conclusions Our results provide detailed insight into the Vif–CBFβ interactions that are critical for Vif function and may contribute to the rational design of HIV-1 inhibitors that block Vif-mediated degradation of A3 proteins. Electronic supplementary material The online version of this article (doi:10.1186/s12977-017-0346-5) contains supplementary material, which is available to authorized users.