Interaction with 7SL RNA but not with HIV-1 genomic RNA or P bodies is required for APOBEC3F virion packaging

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.     

Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.     

7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.     

Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.     

APOBEC3G multimers are recruited to the plasma membrane for packaging into human immunodeficiency virus type 1 virus-like particles in an RNA-dependent process requiring the NC basic linker

APOBEC3G is an endogenous host restriction factor that inhibits human immunodeficiency virus (HIV) replication. The antiviral activity of APOBEC3G is dependent upon its incorporation into the virus particle. The mechanisms governing incorporation of APOBEC3G into virus particles are not completely understood. In particular, some investigators have reported that APOBEC3G interacts directly with the nucleocapsid (NC) subunit of Gag, while others have found that an RNA intermediate is required for Gag-APOBEC3G interactions. In this study, we confirmed the RNA dependence of APOBEC3G packaging and performed detailed mapping of the determinants within NC that are required for virion incorporation. Surprisingly, APOBEC3G packaging did not correlate well with the presence of the N-terminal "I," or interaction, domain within NC. Specifically, Gag constructs containing only the N-terminal region of NC packaged minimal amounts of APOBEC3G, while significant levels of APOBEC3G packaging were achieved with Gag constructs containing the basic linker region of NC. Furthermore, membrane-binding experiments revealed that the basic linker region was essential for the membrane association of APOBEC3G in a Gag-APOBEC3G complex. Fluorescence resonance energy transfer was detected between labeled APOBEC3G in cells and in particles, indicating that APOBEC3G is packaged as a multimer that is bound to packaged RNA. Regions of APOBEC3G-Gag colocalization at the plasma membrane were detected that were distinct from the punctate cytoplasmic bodies where APOBEC3G accumulates within the cell. Together, our results indicate that APOBEC3G multimerizes in an RNA-dependent fashion and that RNA-APOBEC3G multimers are recruited to the plasma membrane and subsequently into virion particles by Gag.     

Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes

APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5′-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.     

Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.     

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.     

Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses.     

HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation

The viral infectivity factor (Vif) encoded by HIV-1 neutralizes a potent antiviral pathway that occurs in human T lymphocytes and several leukemic T-cell lines termed nonpermissive, but not in other cells termed permissive. In the absence of Vif, this antiviral pathway efficiently inactivates HIV-1. It was recently reported that APOBEC3G (also known as CEM-15), a cytidine deaminase nucleic acid-editing enzyme, confers this antiviral phenotype on permissive cells. Here we describe evidence that Vif binds APOBEC3G and induces its rapid degradation, thus eliminating it from cells and preventing its incorporation into HIV-1 virions. Studies of Vif mutants imply that it contains two domains, one that binds APOBEC3G and another with a conserved SLQ(Y/F)LA motif that mediates APOBEC3G degradation by a proteasome-dependent pathway. These results provide promising approaches for drug discovery.     

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.     

RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function.     

Human immunodeficiency virus type 1 replication and regulation of APOBEC3G by peptidyl prolyl isomerase Pin1

APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) replication. HIV-1 synthesizes a viral infectivity factor (Vif) to counter A3G restriction. Currently, it is poorly understood how A3G expression/activity is regulated by cellular factors. Here, we show that the prolyl isomerase Pin1 protein modulates A3G expression. Pin1 was found to be an A3G-interacting protein that reduces A3G expression and its incorporation into HIV-1 virion, thereby limiting A3G-mediated restriction of HIV-1. Intriguingly, HIV-1 infection modulates the phosphorylation state of Pin1, enhancing its ability to moderate A3G activity. These new findings suggest a potential Vif-independent way for HIV-1 to moderate the cellular action of A3G.