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.     

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.     

Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant

Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.     

Monomeric APOBEC3G is catalytically active and has antiviral activity

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.     

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.     

Dimeric RNA Recognition Regulates HIV-1 Genome Packaging

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.     

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.     

Mechanisms of human immunodeficiency virus type 2 RNA packaging: efficient trans packaging and selection of RNA copackaging partners

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.     

Association of potent human antiviral cytidine deaminases with 7SL RNA and viral RNP in HIV-1 virions

7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and tRNA(3)(Lys). Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.     

Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA.

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.     

Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.     

Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis

HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ～10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis- and trans-acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.     

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.