APOBEC3B Potently Restricts HIV-2 but Not HIV-1 in a Vif-Dependent Manner.

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

Comprehensive Investigation on the Interplay between Feline APOBEC3Z3 Proteins and Feline Immunodeficiency Virus Vif Proteins.

As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses. IMPORTANCE Most of the emergences of new virus infections originate from the cross-species transmission of viruses. The fact that some virus infections are strictly specific for the host species indicates that certain "species barriers" in the hosts restrict cross-species jump of viruses, while viruses have evolutionary acquired their own "arms" to overcome/antagonize/neutralize these hurdles. Therefore, understanding of the molecular mechanism leading to successful cross-species viral transmission is crucial for considering the menus of the emergence of novel pathogenic viruses. In the field of retrovirology, APOBEC3-Vif interaction is a well-studied example of the battles between hosts and viruses. Here, we determined the sequences of 11 novel feline APOBEC3Z3 genes and demonstrated that all 18 different feline APOBEC3Z3 proteins tested exhibit anti-feline immunodeficiency virus (FIV) activity. Our comprehensive investigation focusing on the interplay between feline APOBEC3 and FIV Vif can be a clue to elucidate the scenarios of the cross-species transmissions of FIVs in felids.

HIV-2 Vif and foamy virus Bet antagonize APOBEC3B by different mechanisms.

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.

Therapeutic vaccine-mediated Gag-specific CD8+ T-cell induction under anti-retroviral therapy augments anti-virus efficacy of CD8+ cells in simian immunodeficiency virus-infected macaques.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Long-term follow-up and safety assessment of angiogenic gene therapy trial VIF-CAD: Transcatheter intramyocardial administration of a bicistronic plasmid expressing VEGF-A165/bFGF cDNA for the treatment of refractory coronary artery disease.

There have been a number of angiogenic gene therapy trials, yielding mixed results as to efficacy, but demonstrating uniform short-term treatment safety. Data regarding long-term safety of angiogenic gene therapy are limited. Double-blind VIF-CAD trial (NCT00620217) assessed myocardial perfusion and clinical data in 52 refractory coronary artery disease (CAD) patients randomized into treatment (VIF; n = 33) and Placebo (n = 19) arms. VIF group received electromechanical system NOGA-guided intramyocardial injections of VEGF-A165/bFGF plasmid (VIF) into ischemic regions, while the Placebo group-placebo plasmid injections. Full 1-year follow-up data have been published. This study presents the results of over 10-year (median 133 months, range 95-149) safety follow-up of VIF-CAD patients. Overall, 12 (36.4%) patients died in VIF and 8 (42.1%) in Placebo group (P = .68). Cardiovascular mortality was 12/33 (36.4%) in the VIF group and 6/19 (31.6%) in Placebo group (P = .73). Two Placebo patients died due to malignancies, but no VIF patients (P = .17). The Kaplan-Meier curves of combined endpoint: cardiovascular mortality, myocardial infarction and stroke were similar for both patient groups (P = .71). Odds ratio of Placebo group increasing (reaching a worse) their CCS class versus VIF was non-significant (OR 1.28, 95% CI = 0.66-2.45; P = .47). However, CCS class improved in time irrespectively of treatment-OR of reaching a less favorable CCS class per each year of follow-up was 0.74 (95% CI 0.685-0.792; P < .0001, pooled data). There were no differences in readmission rates. Intramyocardial VEGF-A165/bFGF plasmid administration appears safe, with no evidence of an increase in the incidence of death, malignancy, myocardial infarction or stroke during 10-year follow-up in this limited patient population.

The Frequency of Vaccine-Induced T-Cell Responses Does Not Predict the Rate of Acquisition after Repeated Intrarectal SIVmac239 Challenges in Mamu-B*08+ Rhesus Macaques.

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+ ) RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

Mamu-B*17+ Rhesus Macaques Vaccinated with env, vif, and nef Manifest Early Control of SIVmac239 Replication.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ and 50% of Mamu-B*08+ RMs control chronic-phase viremia after SIVmac239 infection. Because CD8+ T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08+ RMs, we investigated whether this might also be true for Mamu-B*17+ RMs. Two groups of Mamu-B*17+ RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08+ RMs, preexisting SIV-specific CD8+ T cells alone do not facilitate long-term virologic suppression in Mamu-B*17+ RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17+ RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection.IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8+ T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17+ macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8+ T cells for virologic control in Mamu-B*17+ macaques and implicate anti-Env antibodies in containment of SIV infection.

Absence of A3Z3-Related Hypermutations in the env and vif Proviral Genes in FIV Naturally Infected Cats.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) proteins comprise an important family of restriction factors that produce hypermutations on proviral DNA and are able to limit virus replication. Vif, an accessory protein present in almost all lentiviruses, counteracts the antiviral A3 activity. Seven haplotypes of APOBEC3Z3 (A3Z3) were described in domestic cats (hap I⁻VII), and in-vitro studies have demonstrated that these proteins reduce infectivity of vif-defective feline immunodeficiency virus (FIV). Moreover, hap V is resistant to vif-mediated degradation. However, studies on the effect of A3Z3 in FIV-infected cats have not been developed. Here, the correlation between APOBEC A3Z3 haplotypes in domestic cats and the frequency of hypermutations in the FIV vif and env genes were assessed in a retrospective cohort study with 30 blood samples collected between 2012 and 2016 from naturally FIV-infected cats in Brazil. The vif and env sequences were analyzed and displayed low or undetectable levels of hypermutations, and could not be associated with any specific A3Z3 haplotype.

Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential.

BACKGROUND: Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication. RESULTS: We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV. CONCLUSIONS: Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.

New World feline APOBEC3 potently controls inter-genus lentiviral transmission.

BACKGROUND: The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) gene family appears only in mammalian genomes. Some A3 proteins can be incorporated into progeny virions and inhibit lentiviral replication. In turn, the lentiviral viral infectivity factor (Vif) counteracts the A3-mediated antiviral effect by degrading A3 proteins. Recent investigations have suggested that lentiviral vif genes evolved to combat mammalian APOBEC3 proteins, and have further proposed that the Vif-A3 interaction may help determine the co-evolutionary history of cross-species lentiviral transmission in mammals. RESULTS: Here we address the co-evolutionary relationship between two New World felids, the puma (Puma concolor) and the bobcat (Lynx rufus), and their lentiviruses, which are designated puma lentiviruses (PLVs). We demonstrate that PLV-A Vif counteracts the antiviral action of APOBEC3Z3 (A3Z3) of both puma and bobcat, whereas PLV-B Vif counteracts only puma A3Z3. The species specificity of PLV-B Vif is irrespective of the phylogenic relationships of feline species in the genera Puma, Lynx and Acinonyx. We reveal that the amino acid at position 178 in the puma and bobcat A3Z3 is exposed on the protein surface and determines the sensitivity to PLV-B Vif-mediated degradation. Moreover, although both the puma and bobcat A3Z3 genes are polymorphic, their sensitivity/resistance to PLV Vif-mediated degradation is conserved. CONCLUSIONS: To the best of our knowledge, this is the first study suggesting that the host A3 protein potently controls inter-genus lentiviral transmission. Our findings provide the first evidence suggesting that the co-evolutionary arms race between lentiviruses and mammals has occurred in the New World.

Feline APOBEC3s, Barriers to Cross-Species Transmission of FIV?

The replication of lentiviruses highly depends on host cellular factors, which defines their species-specific tropism. Cellular restriction factors that can inhibit lentiviral replication were recently identified. Feline immunodeficiency virus (FIV) was found to be sensitive to several feline cellular restriction factors, such as apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) and tetherin, but FIV evolved to counteract them. Here, we describe the molecular mechanisms by which feline APOBEC3 restriction factors inhibit FIV replication and discuss the molecular interaction of APOBEC3 proteins with the viral antagonizing protein Vif. We speculate that feline APOBEC3 proteins could explain some of the observed FIV cross-species transmissions described in wild Felids.

Clues for two-step virion infectivity factor regulation by core binding factor beta.

Lentiviruses threaten human and animal health. Virion infectivity factor (Vif) is essential for the infectivity of most lentiviruses, except for the equine infectious anaemia virus (EIAV). Vif promotes viral infectivity by recruiting a Cullin-based E3 ligase to induce the degradation of a class of host restriction factors, named APOBEC3. Core binding factor beta (CBF-ß) is necessary for several primate lentiviral Vif functions, including HIV-1 Vif. Although much progress has been made in understanding the contribution of CBF-ß to Vif function, the precise mechanism has not yet been fully elucidated. In this study, we found that an interaction with CBF-ß altered the oligomerization and subcellular distribution pattern and increased the stability of two primate lentiviral Vifs, HIV-1 Vif and Macaca simian immunodeficiency virus (SIVmac) Vif. Moreover, using a CBF-ß loss-of-function mutant, we demonstrated that the interaction between CBF-ß and Vif was not sufficient for Vif assistance; a region including F68 in CBF-ß was also required for the stability and function of Vif. For the first time, this study separates the binding and regulating processes of CBF-ß when it is promoting Vif function, which further extends our understanding of the biochemical regulation of Vif by CBF-ß.

Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease.IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals.

Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3.

Species-specific differences in the ability of feline lentiviral Vif to degrade feline APOBEC3 proteins.

How host-virus co-evolutionary relationships manifest is one of the most intriguing issues in virology. To address this topic, the mammal-lentivirus relationship can be considered as an interplay of cellular and viral proteins, particularly apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) and viral infectivity factor (Vif). APOBEC3s enzymatically restrict lentivirus replication, whereas Vif antagonizes the host anti-viral action mediated by APOBEC3. In this study, the focus was on the interplay between feline APOBEC3 proteins and two feline immunodeficiency viruses in cats and pumas. To our knowledge, this study provides the first evidence of non-primate lentiviral Vif being incapable of counteracting a natural host's anti-viral activity mediated via APOBEC3 protein.

A Naturally Occurring Domestic Cat APOBEC3 Variant Confers Resistance to Feline Immunodeficiency Virus Infection.

UNLABELLED: Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary "arms race" between the domestic cat and its cognate lentivirus. IMPORTANCE: Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian immunodeficiency virus [SIV]) if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat, which was likely influenced by lentiviral infection.

Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.

Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

Core-binding factor subunit beta is not required for non-primate lentiviral Vif-mediated APOBEC3 degradation.

Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-ß) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-ß is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-ß. In addition, CBF-ß did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-ß silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-ß was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-ß knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-ß to degrade APOBEC3. CBF-ß did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif-CBF-ß interaction.

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

UNLABELLED: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE: Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.