Vulnerability to APOBEC3G linked to the pathogenicity of deltaretroviruses.

Human retroviruses are derived from simian ones through cross-species transmission. These retroviruses are associated with little pathogenicity in their natural hosts, but in humans, HIV causes AIDS, and human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia-lymphoma (ATL). We analyzed the proviral sequences of HTLV-1, HTLV-2, and simian T-cell leukemia virus type 1 (STLV-1) from Japanese macaques (Macaca fuscata) and found that APOBEC3G (A3G) frequently generates G-to-A mutations in the HTLV-1 provirus, whereas such mutations are rare in the HTLV-2 and STLV-1 proviruses. Therefore, we investigated the mechanism of how HTLV-2 is resistant to human A3G (hA3G). HTLV-1, HTLV-2, and STLV-1 encode the so-called antisense proteins, HTLV-1 bZIP factor (HBZ), Antisense protein of HTLV-2 (APH-2), and STLV-1 bZIP factor (SBZ), respectively. APH-2 efficiently inhibits the deaminase activity of both hA3G and simian A3G (sA3G). HBZ and SBZ strongly suppress sA3G activity but only weakly inhibit hA3G, suggesting that HTLV-1 is incompletely adapted to humans. Unexpectedly, hA3G augments the activation of the transforming growth factor (TGF)-ß/Smad pathway by HBZ, and this activation is associated with ATL cell proliferation by up-regulating BATF3/IRF4 and MYC. In contrast, the combination of APH-2 and hA3G, or the combination of SBZ and sA3G, does not enhance the TGF-ß/Smad pathway. Thus, HTLV-1 is vulnerable to hA3G but utilizes it to promote the proliferation of infected cells via the activation of the TGF-ß/Smad pathway. Antisense factors in each virus, differently adapted to control host cellular functions through A3G, seem to dictate the pathogenesis.

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

Antithetic effect of interferon-α on cell-free and cell-to-cell HIV-1 infection.

In HIV-1-infected individuals, transmitted/founder (TF) virus contributes to establish new infection and expands during the acute phase of infection, while chronic control (CC) virus emerges during the chronic phase of infection. TF viruses are more resistant to interferon-alpha (IFN-α)-mediated antiviral effects than CC virus, however, its virological relevance in infected individuals remains unclear. Here we perform an experimental-mathematical investigation and reveal that IFN-α strongly inhibits cell-to-cell infection by CC virus but only weakly affects that by TF virus. Surprisingly, IFN-α enhances cell-free infection of HIV-1, particularly that of CC virus, in a virus-cell density-dependent manner. We further demonstrate that LY6E, an IFN-stimulated gene, can contribute to the density-dependent enhancement of cell-free HIV-1 infection. Altogether, our findings suggest that the major difference between TF and CC viruses can be explained by their resistance to IFN-α-mediated inhibition of cell-to-cell infection and their sensitivity to IFN-α-mediated enhancement of cell-free infection.

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.

CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE).We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent.Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

A role for gorilla APOBEC3G in shaping lentivirus evolution including transmission to humans.

The APOBEC3 deaminases are potent inhibitors of virus replication and barriers to cross-species transmission. For simian immunodeficiency virus (SIV) to transmit to a new primate host, as happened multiple times to seed the ongoing HIV-1 epidemic, the viral infectivity factor (Vif) must be capable of neutralizing the APOBEC3 enzymes of the new host. Although much is known about current interactions of HIV-1 Vif and human APOBEC3s, the evolutionary changes in SIV Vif required for transmission from chimpanzees to gorillas and ultimately to humans are poorly understood. Here, we demonstrate that gorilla APOBEC3G is a factor with the potential to hamper SIV transmission from chimpanzees to gorillas. Gain-of-function experiments using SIVcpzPtt Vif revealed that this barrier could be overcome by a single Vif acidic amino acid substitution (M16E). Moreover, degradation of gorilla APOBEC3F is induced by Vif through a mechanism that is distinct from that of human APOBEC3F. Thus, our findings identify virus adaptations in gorillas that preceded and may have facilitated transmission to humans.

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool.

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4+ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.

M-Sec facilitates intercellular transmission of HIV-1 through multiple mechanisms.

BACKGROUND: HIV-1 promotes the formation of tunneling nanotubes (TNTs) that connect distant cells, aiding cell-to-cell viral transmission between macrophages. Our recent study suggests that the cellular protein M-Sec plays a role in these processes. However, the timing, mechanism, and to what extent M-Sec contributes to HIV-1 transmission is not fully understood, and the lack of a cell line model that mimics macrophages has hindered in-depth analysis. RESULTS: We found that HIV-1 increased the number, length and thickness of TNTs in a manner dependent on its pathogenic protein Nef and M-Sec in U87 cells, as observed in macrophages. In addition, we found that M-Sec was required not only for TNT formation but also motility of U87 cells, both of which are beneficial for viral transmission. In fact, M-Sec knockdown in U87 cells led to a significantly delayed viral production in both cellular and extracellular fractions. This inhibition was observed for wild-type virus, but not for a mutant virus lacking Nef, which is known to promote not only TNT formation but also migration of infected macrophages. CONCLUSIONS: By taking advantage of useful features of U87 cells, we provided evidence that M-Sec mediates a rapid and efficient cell-cell transmission of HIV-1 at an early phase of infection by enhancing both TNT formation and cell motility.

Quantifying the antiviral effect of APOBEC3 on HIV-1 infection in humanized mouse model.

APOBEC3 proteins inhibit human immunodeficiency virus (HIV)-1 infection by independently impairing viral reverse transcription and inducing G-to-A mutations in viral DNA. An HIV-1-encoded protein, viral infectivity factor (Vif), can counteract these antiviral activities of APOBEC3 proteins. Although previous studies using in vitro cell culture systems have revealed the molecular mechanisms of the antiviral action of APOBEC3 proteins and their antagonism by Vif, it remains unclear how APOBEC3 proteins affect the kinetics of HIV-1 replication in vivo. Here we quantified the time-series of viral load datasets from humanized mice infected with HIV-1 variants in the presence of APOBEC3F, APOBEC3G, or both APOBEC3F/G using a simple mathematical model that accounted for inter-individual variability. Through experimental and mathematical investigation, we formulated and calculated the total antiviral activity of APOBEC3F and APOBEC3G based on the estimated initial growth rates of viral loads in vivo. Interestingly, we quantitatively demonstrated that compared with APOBEC3G, the antiviral activity of APOBEC3F was widely distributed but skewed toward lower activity, although their mean values were similar. We concluded that APOBEC3G markedly and robustly restricted the initial stages of viral growth in vivo. This is the first report to quantitatively elucidate how APOBEC3F and APOBEC3G differ in their anti-HIV-1 modes in vivo.

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.

Correction: HIV-1 competition experiments in humanized mice show that APOBEC3H imposes selective pressure and promotes virus adaptation.

[This corrects the article DOI: 10.1371/journal.ppat.1006348.].

HIV-1 competition experiments in humanized mice show that APOBEC3H imposes selective pressure and promotes virus adaptation.

APOBEC3 (A3) family proteins are DNA cytosine deaminases recognized for contributing to HIV-1 restriction and mutation. Prior studies have demonstrated that A3D, A3F, and A3G enzymes elicit a robust anti-HIV-1 effect in cell cultures and in humanized mouse models. Human A3H is polymorphic and can be categorized into three phenotypes: stable, intermediate, and unstable. However, the anti-viral effect of endogenous A3H in vivo has yet to be examined. Here we utilize a hematopoietic stem cell-transplanted humanized mouse model and demonstrate that stable A3H robustly affects HIV-1 fitness in vivo. In contrast, the selection pressure mediated by intermediate A3H is relaxed. Intriguingly, viral genomic RNA sequencing reveled that HIV-1 frequently adapts to better counteract stable A3H during replication in humanized mice. Molecular phylogenetic analyses and mathematical modeling suggest that stable A3H may be a critical factor in human-to-human viral transmission. Taken together, this study provides evidence that stable variants of A3H impose selective pressure on HIV-1.

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.

Different effects of two mutations on the infectivity of Ebola virus glycoprotein in nine mammalian species.

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann-Pick C1.

A naturally occurring feline APOBEC3 variant that loses anti-lentiviral activity by lacking two amino acid residues.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) is a mammalian protein that restricts lentiviral replication. Various polymorphisms of mammalian APOBEC3 genes have been observed in humans, Old World monkeys and domestic cats; however, the genetic diversity of APOBEC3 genes in other mammals remains unaddressed. Here we identify a novel haplotype of the feline APOBEC3Z3 gene, an APOBEC3 gene that restricts feline immunodeficiency virus (FIV) replication, in a Eurasian lynx (Lynx lynx). Compared to the previously identified lynx APOBEC3Z3 (haplotype I), the new sequence (haplotype II) harbours two amino acid deletions (Q16 and H17) and a nonsynonymous substitution (R68Q). Interestingly, lynx APOBEC3Z3 haplotype II does not suppress FIV infectivity, whereas haplotype I does. Mutagenesis experiments further revealed that deleting two amino acids (Q16 and H17) causes anti-FIV activity loss. This report demonstrates that a naturally occurring APOBEC3 variant loses anti-lentiviral activity through the deletion of two amino acid residues.

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.

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.

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

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.

A conflict of interest: the evolutionary arms race between mammalian APOBEC3 and lentiviral Vif.

Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are mammalian-specific cellular deaminases and have a robust ability to restrain lentivirus replication. To antagonize APOBEC3-mediated antiviral action, lentiviruses have acquired viral infectivity factor (Vif) as an accessory gene. Mammalian APOBEC3 proteins inhibit lentiviral replication by enzymatically inserting G-to-A hypermutations in the viral genome, whereas lentiviral Vif proteins degrade host APOBEC3 via the ubiquitin/proteasome-dependent pathway. Recent investigations provide evidence that lentiviral vif genes evolved to combat mammalian APOBEC3 proteins. In corollary, mammalian APOBEC3 genes are under Darwinian selective pressure to escape from antagonism by Vif. Based on these observations, it is widely accepted that lentiviral Vif and mammalian APOBEC3 have co-evolved and this concept is called an "evolutionary arms race." This review provides a comprehensive summary of current knowledge with respect to the evolutionary dynamics occurring at this pivotal host-virus interface.