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.     

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.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

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.     

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.     

APOBEC3G incorporation into human immunodeficiency virus type 1 particles

APOBEC3G is promiscuous with respect to its antiretroviral effect, requiring that it be packaged into diverse retrovirus particles. Here, we show that most virally encoded human immunodeficiency virus type 1 particle components are dispensable for APOPEC3G incorporation. However, replacement of the nucleocapsid (NC) Gag domain with a leucine zipper abolished APOBEC3G incorporation. Moreover, coprecipitation analysis showed that APOBEC3G-Gag interaction requires NC and nonspecific RNA. These observations suggest that APOBEC3G exploits an essential property of retroviruses, namely, RNA packaging, to infiltrate particles. Because it is, therefore, difficult to evolve specific sequences that confer escape from APOBEC3G, these findings may explain why lentiviruses evolved an activity that induces its destruction.     

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.     

HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.     

HIV-1 Viral Infectivity Factor (Vif) Alters Processive Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (−)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of Vif_IIIB- and Vif_HXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, Vif_HXB2 inhibited the jumping and Vif_IIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.     

Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.     

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.     

Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal

The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.