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.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

[Multifunctional HIV accessory proteins are hub proteins antagonizing host antiviral factors].

HIV has several accessory proteins (Vif, Vpu, Vpr, Vpx, and Nef) along with structural /enzymatic (Gag, Pol, and Env) and gene-expression regulatory proteins (Tat and Rev) essential for viral replication. The accessory proteins are neither required in some kinds of cells and nor all conserved between HIV-1 and HIV-2. For these reasons, their functional roles and mechanisms had been unclear. However, since a finding of Vif's neutralizing function against host restriction factor APOBEC3G, it has been elucidated that the accessory proteins play critical roles to antagonize host intrinsic antiviral activity. So far, in addition to Vif-APOBEC3, Vpu-BST-2/Tetherin and Vpx-SAMHD1 have been identified as such examples. Here, we summarize the biological functions and features on HIV accessory proteins in terms of antagonizing factors against the host antiviral factors.

HIV and SIV infection: the role of cellular restriction and immune responses in viral replication and pathogenesis.

The human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have a long biological history. Both viruses evolved from Africa and remnants of them can be found in the 'fossil record' of several species in which they are not endemic. SIV remains endemic in several species of monkeys in Africa where it does not cause immune deficiency. HIV and SIV actively replicate within humans and Asian non-human primates, despite cellular and genetic viral restriction factors and genes, and at times robust innate and adaptive immune responses. While Lentiviruses are considered 'slow viruses' it is clear in humans and susceptible Asian monkeys that virus production is rapid and highly active. This results in a massive loss of CD4+ memory effector T cells early after infection and a continued race between viral evolution, cytotoxic lymphocytes, and failed neutralizing antibody responses. Concurrently, HIV and SIV can infect monocyte/macrophage populations in blood and more importantly in tissues, including the central nervous system, where the virus can remain sequestered and not cleared by anti-retroviral therapy, and hide for years. This review will discuss species and cellular barriers to infection, and the role of innate and acquired immunity with infection and pathogenesis of HIV and SIV in select species.

Amino acid alterations in Gag that confer the ability to grow in simian cells on HIV-1 are located at a narrow CA region.

We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.

Cytidine deamination induced HIV-1 drug resistance.

The HIV-1 Vif protein is essential for overcoming the antiviral activity of DNA-editing apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) cytidine deaminases. We show that naturally occurring HIV-1 Vif point mutants with suboptimal anti-APOBEC3G activity induce the appearance of proviruses with lamivudine (3TC) drug resistance-associated mutations before any drug exposure. These mutations, ensuing from cytidine deamination events, were detected in >40% of proviruses with partially defective Vif mutants. Transfer of drug resistance from hypermutated proviruses via recombination allowed for 3TC escape under culture conditions prohibitive for any WT viral growth. These results demonstrate that defective hypermutated genomes can shape the phenotype of the circulating viral population. Partially active Vif alleles resulting in incomplete neutralization of cytoplasmic APOBEC3 molecules are directly responsible for the generation of a highly diverse, yet G-to-A biased, proviral reservoir, which can be exploited by HIV-1 to generate viable and drug-resistant progenies.

The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements.

All retroviruses, including HIV-1, display species-specific patterns of infection. The impaired growth of these retroviruses in foreign and sometimes even in their natural hosts often stems from the action of potent host-encoded "viral restriction factors" that form important protective components of the innate immune system. The discovery of APOBEC3G and related cytidine deaminases as one class of host restriction factors and of the action of HIV-1 Vif as a specific APOBEC3G antagonist have stimulated intense scientific interest. This Vif-APOBEC3G axis now forms a very attractive target for development of an entirely new class of anti-HIV drugs. In this review, we summarize current understanding of the mechanism of action of the APOBEC3 family of enzymes, their intriguing regulation within cells, the impact of these enzymes on viral evolution and disease progression, and their roles in controlling not only the replication of exogenous retroviruses but also the retrotransposition of endogenous retroelements.

Vpr.A3A chimera inhibits HIV replication.

Several APOBEC3 proteins (A3F and A3G), that are cytidine deaminases restrict human immunodeficiency virus (HIV) replication in the absence of the viral infectivity factor (Vif) protein. However, Vif leads to their degradation and counteracts their effects. Another member, A3A, restricts some retrotransposons and another virus but not HIV. We reasoned that this failure was due to the lack of appropriate targeting. Thus, we fused A3A to another viral protein, Vpr, which binds p6 in Gag and is incorporated into viral cores. Indeed, the Vpr.A3A chimera but not A3A was found abundantly in the viral core. It also restricted potently the replication of HIV and simian immunodeficiency virus in the presence and absence of Vif. Because we identified a high frequency of G to A mutations in viral cDNAs, this antiviral activity was mediated by DNA editing. Interestingly, our fusion protein did not restrict murine leukemia virus, which does not incorporate Vpr. Thus, by targeting appropriately a potent single domain cytidine deaminase, we rendered HIV and simian immunodeficiency virus restriction resistant to Vif.

Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H.

APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older "mature" A3G already residing in high-molecular-mass RNA-protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.

Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family.

A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.

Redirecting coronavirus to a nonnative receptor through a virus-encoded targeting adapter.

Murine hepatitis coronavirus (MHV)-A59 infection depends on the interaction of its spike (S) protein with the cellular receptor mCEACAM1a present on murine cells. Human cells lack this receptor and are therefore not susceptible to MHV. Specific alleviation of the tropism barrier by redirecting MHV to a tumor-specific receptor could lead to a virus with appealing properties for tumor therapy. To demonstrate that MHV can be retargeted to a nonnative receptor on human cells, we produced bispecific adapter proteins composed of the N-terminal D1 domain of mCEACAM1a linked to a short targeting peptide, the six-amino-acid His tag. Preincubation of MHV with the adapter proteins and subsequent inoculation of human cells expressing an artificial His receptor resulted in infection of these otherwise nonsusceptible cells and led to subsequent production of progeny virus. To generate a self-targeted virus able to establish multiround infection of the target cells, we subsequently incorporated the gene encoding the bispecific adapter protein as an additional expression cassette into the MHV genome through targeted RNA recombination. When inoculated onto murine LR7 cells, the resulting recombinant virus indeed expressed the adapter protein. Furthermore, inoculation of human target cells with the virus resulted in a His receptor-specific infection that was multiround. Extensive cell-cell fusion and rapid cell killing of infected target cells was observed. Our results show that MHV can be genetically redirected via adapters composed of the S protein binding part of mCEACAM1a and a targeting peptide recognizing a nonnative receptor expressed on human cells, consequently leading to rapid cell death. The results provide interesting leads for further investigations of the use of coronaviruses as antitumor agents.

Review of the twelfth West Coast Retrovirus Meeting.

Every year the Cancer Research Institute from University of California at Irvine organizes the West Coast Retrovirus Meeting where participants have a chance to discuss the latest progress in understanding the pathology of retroviruses. The 12th meeting was held at the Hyatt Regency Suites in Palm Springs, California from October 6th to October 9th 2005, with the major focus on human immunodeficiency virus (HIV) pathogenesis. Philippe Gallay from The Scripps Research Institute and Thomas J. Hope from Northwestern University organized the meeting, which covered all the steps involved in the lifecycle of retroviruses with an emphasis on virus:host interactions. The trend in research appeared to be on the restriction of viral infection, both by the endogenous, cellular restriction factors, as well as by the potential antimicrobial compounds of known or unknown mechanisms. Additionally, new stories on the inevitable feedback from the host immune system were presented as well. HIV still represents a challenge that an army of motivated people has been working on for over 20 years. And yet, the field has not reached the plateau in knowledge nor enthusiasm, which was proven again in October 2005 in Palm Springs.

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo.

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.

Complementary function of the two catalytic domains of APOBEC3G.

The HIV-1 viral accessory protein Vif prevents the encapsidation of the antiviral cellular cytidine deaminases APOBEC3F and APOBEC3G by inducing their proteasomal degradation. In the absence of Vif, APOBEC3G is encapsidated and blocks virus replication by deaminating cytosines of the viral cDNA. APOBEC3G encapsidation has been recently shown to depend on the viral nucleocapsid protein; however, the role of RNA remains unclear. Using APOBEC3G deletion and point mutants, we mapped the encapsidation determinant to the Zn(2+) coordination residues of the N-terminal catalytic domain (CD1). Notably, these residues were also required for RNA binding. Mutations in the two aromatic residues of CD1 but not CD2, which are conserved in cytidine deaminase core domains and are required for RNA binding, prevented encapsidation into HIV-1, HTLV-I and MLV. The Zn(2+) coordination residues of the C-terminal catalytic domain (CD2) were not required for encapsidation but were essential for cytidine deaminase activity and the antiviral effect. These findings suggest a model in which CD1 mediates encapsidation and RNA binding while CD2 mediates cytidine deaminase activity. Interestingly, HTLV-I was relatively resistant to the antiviral effects of encapsidated APOBEC3G.

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease.

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).

Further investigation of simian immunodeficiency virus Vif function in human cells.

Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two vif-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.

Vif is an auxiliary factor of the HIV-1 reverse transcriptase and facilitates abasic site bypass.

The HIV-1 accessory protein Vif was found to modulate the RNA- and DNA-dependent DNA synthesis activity of the viral RT (reverse transcriptase) in two ways: (i) it stimulated the binding of the viral RT to the primer by increasing the association rate kcat/K(m) and by decreasing the thermodynamic barrier DeltaH([ES]) for complex formation, and (ii) it increased the polymerization rate of HIV-1 RT. A Vif mutant lacking the final 56 amino acids at the C-terminus failed to stimulate the viral RT. On the other hand, another Vif mutant lacking the first 43 amino acids at the N-terminus, which are involved in RNA binding and interaction with the viral protease, was able to stimulate RT activity. In addition, Vif was found to promote the bypass of an abasic site by HIV-1 RT.

Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication.

Recently, APOBEC3G has been identified as a host factor that blocks retroviral replication. It introduces G to A hypermutations in newly synthesized minus strand viral cDNA at the step of reverse transcription in target cells. Here, we identified the human APOBEC3F protein as another host factor that blocks human immunodeficiency virus type 1 (HIV-1) replication. Similar to APOBEC3G, APOBEC3F also induced G to A hypermutations in HIV genomic DNA, and the viral Vif protein counteracted its activity. Thus, APOBEC family members might have evolved as a general defense mechanism of the body against retroviruses, retrotransposons, and other mobile genetic elements.

The interaction between HIV-1 Gag and APOBEC3G.

APOBEC3G, a member of an RNA/DNA cytidine deaminase superfamily, has been identified as a cellular inhibitor of HIV-1 infectivity, possibly through the dC to dU deamination of the first minus strand cDNA synthesized during reverse transcription. Virions incorporate APOBEC3G during viral assembly in non-permissive cells, and this incorporation is inhibited by the viral protein Vif. The mechanism of APOBEC3G incorporation into HIV-1 is examined in this report. In the absence of Vif, cytoplasmic APOBEC3G becomes membrane-bound in cells expressing HIV-1 Gag, and its incorporation into Gag viral-like particles (VLPs) is proportional to the amount of APOBEC3G expressed in the cell. The expression of Vif, or mutant Gag unable to bind to membrane, prevents the APOBEC3G association with membrane. HIV-1 Gag alone among viral proteins is sufficient for packaging of APOBEC3G into Gag VLPs, and this incorporation requires the presence of Gag nucleocapsid. The presence of amino acids 104-156 in APOBEC3G, located in the linker region between two zinc coordination motifs, is also required for its incorporation into Gag VLPs. Evidence against an RNA bridge facilitating the Gag/APOBEC3G interaction includes data indicating that 1) the incorporation of APOBEC3G occurs independently of viral genomic RNA, 2) a Gag/APOBEC3G complex is immunoprecipitated from cell lysate after RNase treatment, and 3) the zinc coordination motif, rather than the regions flanking this motif, have been implicated in RNA binding in another family member, APOBEC1.

Influence of primate lentiviral Vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of APOBEC3G.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral evasion of the host antiviral protein APOBEC3G, also known as CEM15. Vif mutant but not wild-type HIV-1 viruses produced in the presence of APOBEC3G have been shown to undergo hypermutations in newly synthesized viral DNA upon infection of target cells, presumably resulting from C-to-U modification during minus-strand viral DNA synthesis. We now report that HIV-1 Vif could induce rapid degradation of human APOBEC3G that was blocked by the proteasome inhibitor MG132. The efficiency of Vif-induced downregulation of APOBEC3G expression depended on the level of Vif expression. A single amino acid substitution in the conserved SLQXLA motif reduced Vif function. Vif proteins from distantly related primate lentiviruses such as SIVagm were unable to suppress the antiviral activity of human APOBEC3G or the packaging of APOBEC3G into HIV-1 Vif mutant virions, due to a lack of interaction with human APOBEC3G. In the presence of the proteasome inhibitor MG132, virion-associated Vif increased dramatically. However, increased virion packaging of Vif did not prevent virion packaging of APOBEC3G when proteasome function was impaired, and the infectivity of these virions was significantly reduced. These results suggest that Vif function is required during virus assembly to remove APOBEC3G from packaging into released virions. Once packaged, virion-associated Vif could not efficiently block the antiviral activity of APOBEC3G.