Evaluating HIV-1 Infectivity and Virion Maturation Across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRETΔEnv) derived from Jurkat (a human T lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposi's sarcoma herpesvirus (KSHV) ORF34 is a component of the viral pre-initiation complex (vPIC), a highly conserved piece of machinery essential for late gene expression among beta- and gamma-herpes viruses. KSHV ORF34 is also estimated to be a hub protein, associated with the majority of vPIC components. However, the precise mechanisms underlying how the ORF34 molecule contributes to the vPIC function, including the binding manner to other vPIC components, remain unclear. Therefore, we constructed ORF34 alanine-scanning mutants, in which amino-acid residues that were conserved among other herpesviruses had been replaced by alanine. The mutants were analyzed for their binding functions to other vPIC factors, and then were evaluated for their recovering ability of viral production using the cells harboring ORF34-deficient KSHV-BAC. The results demonstrated that at least four cysteines conserved in ORF34 were crucial for binding to other vPIC components, ORF24 and ORF66, virus production, and late gene transcription and expression. Based on the amino acid sequence of ORF34, these four cysteines were expected to constitute a pair of C-Xn-C consensus motifs. An artificial intelligence-predicted structure model revealed that the four cysteines were present tetrahedrally in an intramolecular fashion. Another prediction algorithm indicated the possible capture of metal cations by ORF34. Furthermore, it was experimentally observed that the elimination of cations by a selective chelator resulted in the loss of ORF34's binding ability to other vPIC components. In conclusion, our results suggest the functional importance of KSHV ORF34 conserved cysteines for vPIC components assembly and viral replication.

Biomolecular Fluorescence Complementation Profiling and Artificial Intelligence Structure Prediction of the Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Interaction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the protein-protein interactions of the vPIC factors have not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral proteins has not been sufficiently revealed. In order to clarify these issues, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Glu36, His41, and Trp170) of ORF18 that were responsible for its interaction with ORF30. Pull-down assays also showed that these four residues were required for the ORF18-ORF30 interaction. The artificial intelligence (AI) system AlphaFold2 predicted that the identified four residues are localized on the surface of ORF18 and are in proximity to each other. Thus, our AI-predicted model supports the importance of the four residues for binding ORF18 to ORF30. These results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.

Insights Into Persistent HIV-1 Infection and Functional Cure: Novel Capabilities and Strategies.

Although HIV-1 replication can be efficiently suppressed to undetectable levels in peripheral blood by combination antiretroviral therapy (cART), lifelong medication is still required in people living with HIV (PLWH). Life expectancies have been extended by cART, but age-related comorbidities have increased which are associated with heavy physiological and economic burdens on PLWH. The obstacle to a functional HIV cure can be ascribed to the formation of latent reservoir establishment at the time of acute infection that persists during cART. Recent studies suggest that some HIV reservoirs are established in the early acute stages of HIV infection within multiple immune cells that are gradually shaped by various host and viral mechanisms and may undergo clonal expansion. Early cART initiation has been shown to reduce the reservoir size in HIV-infected individuals. Memory CD4+ T cell subsets are regarded as the predominant cellular compartment of the HIV reservoir, but monocytes and derivative macrophages or dendritic cells also play a role in the persistent virus infection. HIV latency is regulated at multiple molecular levels in transcriptional and post-transcriptional processes. Epigenetic regulation of the proviral promoter can profoundly regulate the viral transcription. In addition, transcriptional elongation, RNA splicing, and nuclear export pathways are also involved in maintaining HIV latency. Although most proviruses contain large internal deletions, some defective proviruses may induce immune activation by expressing viral proteins or producing replication-defective viral-like particles. In this review article, we discuss the state of the art on mechanisms of virus persistence in the periphery and tissue and summarize interdisciplinary approaches toward a functional HIV cure, including novel capabilities and strategies to measure and eliminate the infected reservoirs and induce immune control.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Effect of P-body component Mov10 on HCV virus production and infectivity.

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.

Defective HIV-1 proviruses produce viral proteins.

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34.

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV-encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late (L) gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the viral preinitiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, -24, -30, -31, -34, and -66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a bacterial artificial chromosome (BAC) system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased virus production, viral DNA replication was unaffected. Chromatin immunoprecipitation analysis showed that ORF66 bound to the promoters of a late gene (K8.1) but did not bind to those of a latent gene (ORF72), an immediate early gene (ORF16), or an early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine repeat in the C-terminal region of ORF66 were essential for the interaction with ORF34, the transcription of K8.1, and virus production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model in which ORF66 serves as a critical vPIC component to promote late viral gene expression and virus production.IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therefore, we generated an ORF66-deficient BAC clone and evaluated its viral replication. The results showed that ORF66 plays a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing the function of ORF66 in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacts with the transcription start site of the K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among beta- and gammaherpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

A vaccine-induced gene expression signature correlates with protection against SIV and HIV in multiple trials.

Current HIV vaccines are only partially efficacious, presenting an opportunity to identify correlates of protection and, thereby, potential insight into mechanisms that prevent HIV acquisition. Two independent preclinical challenge studies in nonhuman primates (NHPs) previously showed partial efficacy of a mosaic adenovirus 26 (Ad26)-based HIV-1 vaccine candidate. To investigate the basis of this protection, we performed whole transcriptomics profiling by RNA sequencing (RNA-seq) in sorted lymphocytes from peripheral blood samples taken during these studies at different time points after vaccination but before challenge. We observed a transcriptional signature in B cells that associated with protection from acquisition of simian immunodeficiency virus (SIV) or the simian-human immunodeficiency virus (SHIV) in both studies. Strong antibody responses were elicited, and genes from the signature for which expression was enriched specifically associated with higher magnitude of functional antibody responses. The same gene expression signature also associated with protection in RV144 in the only human HIV vaccine trial to date that has shown efficacy and in two additional NHP studies that evaluated similar canarypox-based vaccine regimens. A composite gene expression score derived from the gene signature was one of the top-ranked correlates of protection in the NHP vaccine studies. This study aims to bridge preclinical and clinical data with the identification of a gene signature in B cells that is associated with protection from SIV and HIV infection by providing a new approach for evaluating future vaccine candidates.

Structural Determinants of the APOBEC3G N-Terminal Domain for HIV-1 RNA Association.

APOBEC3G (A3G) is a cellular protein that inhibits HIV-1 infection through virion incorporation. The interaction of the A3G N-terminal domain (NTD) with RNA is essential for A3G incorporation in the HIV-1 virion. The interaction between A3G-NTD and RNA is not completely understood. The A3G-NTD is also recognized by HIV-1 Viral infectivity factor (Vif) and A3G-Vif binding leads to A3G degradation. Therefore, the A3G-Vif interaction is a target for the development of antiviral therapies that block HIV-1 replication. However, targeting the A3G-Vif interactions could disrupt the A3G-RNA interactions that are required for A3G's antiviral activity. To better understand A3G-RNA binding, we generated in silico docking models to simulate the RNA-binding propensity of A3G-NTD. We simulated the A3G-NTD residues with high RNA-binding propensity, experimentally validated our prediction by testing A3G-NTD mutations, and identified structural determinants of A3G-RNA binding. In addition, we found a novel amino acid residue, I26 responsible for RNA interaction. The new structural insights provided here will facilitate the design of pharmaceuticals that inhibit A3G-Vif interactions without negatively impacting A3G-RNA interactions.

Experimental Adaptive Evolution of Simian Immunodeficiency Virus SIVcpz to Pandemic Human Immunodeficiency Virus Type 1 by Using a Humanized Mouse Model.

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, originated from simian immunodeficiency virus from chimpanzees (SIVcpz), the precursor of the human virus, approximately 100 years ago. This indicates that HIV-1 has emerged through the cross-species transmission of SIVcpz from chimpanzees to humans. However, it remains unclear how SIVcpz has evolved into pandemic HIV-1 in humans. To address this question, we inoculated three SIVcpz strains (MB897, EK505, and MT145), four pandemic HIV-1 strains (NL4-3, NLCSFV3, JRCSF, and AD8), and two nonpandemic HIV-1 strains (YBF30 and DJO0131). Humanized mice infected with SIVcpz strain MB897, a virus phylogenetically similar to pandemic HIV-1, exhibited a peak viral load comparable to that of mice infected with pandemic HIV-1, while peak viral loads of mice infected with SIVcpz strain EK505 or MT145 as well as nonpandemic HIV-1 strains were significantly lower. These results suggest that SIVcpz strain MB897 is preadapted to humans, unlike the other SIVcpz strains. Moreover, viral RNA sequencing of MB897-infected humanized mice identified a nonsynonymous mutation in env, a G413R substitution in gp120. The infectivity of the gp120 G413R mutant of MB897 was significantly higher than that of parental MB897. Furthermore, we demonstrated that the gp120 G413R mutant of MB897 augments the capacity for viral replication in both in vitro cell cultures and humanized mice. Taken together, this is the first experimental investigation to use an animal model to demonstrate a gain-of-function evolution of SIVcpz into pandemic HIV-1.IMPORTANCE From the mid-20th century, humans have been exposed to the menace of infectious viral diseases, such as severe acute respiratory syndrome coronavirus, Ebola virus, and Zika virus. These outbreaks of emerging/reemerging viruses can be triggered by cross-species viral transmission from wild animals to humans, or zoonoses. HIV-1, the causative agent of AIDS, emerged by the cross-species transmission of SIVcpz, the HIV-1 precursor in chimpanzees, around 100 years ago. However, the process by which SIVcpz evolved to become HIV-1 in humans remains unclear. Here, by using a hematopoietic stem cell-transplanted humanized-mouse model, we experimentally recapitulate the evolutionary process of SIVcpz to become HIV-1. We provide evidence suggesting that a strain of SIVcpz, MB897, preadapted to infect humans over other SIVcpz strains. We further demonstrate a gain-of-function evolution of SIVcpz in infected humanized mice. Our study reveals that pandemic HIV-1 has emerged through at least two steps: preadaptation and subsequent gain-of-function mutations.

Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency.

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence.

A naturally occurring bovine APOBEC3 confers resistance to bovine lentiviruses: implication for the co-evolution of bovids and their lentiviruses.

Mammals have co-evolved with lentiviruses for a long time. As evidence, viral infectivity factor (Vif), encoded by lentiviruses, antagonizes the anti-viral action of cellular APOBEC3 of their hosts. Here, we address the co-evolutionary dynamics of bovine APOBEC3 and the following two bovine lentiviruses: bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV). We determined the sequences of three APOBEC3 genes of bovids belonging to the genera Bos and Bison and showed that bovine APOBEC3Z3 is under a strong positive selection. We found that APOBEC3Z3 of gaur, a bovid in the genus Bos, acquired resistance to JDV Vif-mediated degradation after diverging from the other bovids through conversion of the structural composition of the loop 1 domain. Interestingly, the resistance of gaur APOBEC3Z3 can be attributed to the positive selection of residue 62. This study provides the first evidence, suggesting that a co-evolutionary arms race between bovids and lentiviruses occurred in Asia.

APOBEC3 proteins can copackage and comutate HIV-1 genomes.

Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence of A3G + A3F and A3G + A3H, exhibited extensive comutation of viral cDNA, as determined by the frequency of G-to-A mutations in the proviral genomes in the contexts of A3G (GG-to-AG) and A3D, A3F or A3H (GA-to-AA) edited sites. The copackaging of A3G + A3F and A3G + A3H resulted in an additive increase and a modest synergistic increase (1.8-fold) in the frequency of GA-to-AA mutations, respectively. We also identified distinct editing site trinucleotide sequence contexts for each APOBEC3 protein and used them to show that hypermutation of proviral DNAs from seven patients was induced by A3G, A3F (or A3H), A3D and A3G + A3F (or A3H). These results indicate that APOBEC3 proteins can be copackaged and can comutate the same genomes, and can cooperate to inhibit HIV replication.

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.

Coevolutionary dynamics between tribe Cercopithecini tetherins and their lentiviruses.

Human immunodeficiency virus, a primate lentivirus (PLV), causes AIDS in humans, whereas most PLVs are less or not pathogenic in monkeys. These notions suggest that the co-evolutionary process of PLVs and their hosts associates with viral pathogenicity, and therefore, that elucidating the history of virus-host co-evolution is one of the most intriguing topics in the field of virology. To address this, recent studies have focused on the interplay between intrinsic anti-viral proteins, such as tetherin, and viral antagonists. Through an experimental-phylogenetic approach, here we investigate the co-evolutionary interplay between tribe Cercopithecini tetherin and viral antagonists, Nef and Vpu. We reveal that tribe Cercopithecini tetherins are positively selected, possibly triggered by ancient Nef-like factor(s). We reconstruct the ancestral sequence of tribe Cercopithecini tetherin and demonstrate that all Nef proteins are capable of antagonizing ancestral Cercopithecini tetherin. Further, we consider the significance of evolutionary arms race between tribe Cercopithecini and their PLVs.

APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.