A G-C-Rich Palindromic Structural Motif and a Stretch of Single-Stranded Purines Are Required for Optimal Packaging of Mason-Pfizer Monkey Virus (MPMV) Genomic RNA

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5′ end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5′ untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region “A”, immediately downstream of the primer binding site, is composed of 50 nt, whereas region “B” is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.     

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78^Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.     

Identification of molecular determinants from Moloney leukemia virus 10 homolog (MOV10) protein for virion packaging and anti-HIV-1 activity

Discovery of novel antiretroviral mechanism is essential for the design of innovative antiretroviral therapy. Recently, we and others reported that ectopic expression of Moloney leukemia virus 10 (MOV10) protein strongly inhibits retrovirus replication. MOV10, a putative RNA helicase, can be packaged into HIV-1 virions by binding to the nucleocapsid (NC) region of Gag and inhibit viral replication at a postentry step. Here, we report critical determinants for MOV10 virion packaging and antiviral activity. MOV10 has 1,003 amino acids and seven helicase motifs. We found that MOV10 packaging requires the NC basic linker, and Gag binds to the N-terminal amino acids 261-305 region of MOV10. Our predicted MOV10 three-dimensional structure model indicates that the Gag binding region is located in a structurally exposed domain, which spans amino acids 93-305 and is Cys-His-rich. Simultaneous mutation of residues Cys-188, Cys-195, His-199, His-201, and His-202 in this domain significantly compromised MOV10 anti-HIV-1 activity. Notably, although MOV10-Gag interaction is required, it is not sufficient for MOV10 packaging, which also requires its C-terminal all but one of seven helicase motifs. Moreover, we have mapped the minimal MOV10 antiviral region to amino acids 99-949, indicating that nearly all MOV10 residues are required for its antiviral activity. Mutations of residues Cys-947, Pro-948, and Phe-949 at the C terminus of this region completely disrupted MOV10 anti-HIV-1 activity. Taken together, we have identified two critical MOV10 packaging determinants and eight other critical residues for anti-HIV-1 activity. These results provide a molecular basis for further understanding the MOV10 antiretroviral mechanism.     

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77^Gag along with in cell gRNA packaging study identified two Pr77^Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77^Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.     

Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.     

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.     

The Gag Domains Required for Avian Retroviral RNA Encapsidation Determined by Using Two Independent Assays

The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity. Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation.     

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55^Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4⁺ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.     

Beyond Plasma Membrane Targeting: Role of the MA domain of Gag in Retroviral Genome Encapsidation

The MA (matrix) domain of the retroviral Gag polyprotein plays several critical roles during virus assembly. Although best known for targeting the Gag polyprotein to the inner leaflet of the plasma membrane for virus budding, recent studies have revealed that MA also contributes to selective packaging of the genomic RNA (gRNA) into virions. In this Review, we summarize recent progress in understanding how MA participates in genome incorporation. We compare the mechanisms by which the MA domains of different retroviral Gag proteins influence gRNA packaging, highlighting variations and similarities in how MA directs the subcellular trafficking of Gag, interacts with host factors and binds to nucleic acids. A deeper understanding of how MA participates in these diverse functions at different stages in the virus assembly pathway will require more detailed information about the structure of the MA domain within the full-length Gag polyprotein. In particular, it will be necessary to understand the structural basis of the interaction of MA with gRNA, host transport factors and membrane phospholipids. A better appreciation of the multiple roles MA plays in genome packaging and Gag localization might guide the development of novel antiviral strategies in the future.     

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5′end of the viral gRNA, known as the packaging signal (ψ), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to ψ, as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Rev-responsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16, 35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17, 18, 37, 41). Simple retroviruses do not encode Rev-like regulatory proteins, so other strategies for the export of unspliced viral RNAs are needed. For Mason-Pfizer monkey virus, a cis-acting constitutive transport element induces nuclear export of the unspliced viral RNA in a process mediated by the cellular mRNA nuclear export factor TAP (5, 25, 46, 63). In RSV, an RNA element composed of either of the two direct repeats flanking the src gene mediates the cytoplasmic accumulation of unspliced viral RNA by using host export proteins TAP and Dpb5 (29, 42, 44).The findings of recent studies suggest that specific RNA export pathways direct viral gRNA to sites of virion assembly (56); for example, HIV-1 gRNA export out of the nucleus by the Rev-RRE-CRM1 complex is required for the proper subcellular localization of Gag and efficient virus particle production (26, 57). In the case of RSV, little is known about the trafficking of the viral RNA destined for virion encapsidation or the effects of the gRNA nuclear export pathway on Gag trafficking and virus particle production. However, we do know that RSV Gag enters the nucleus during infection, owing to nuclear localization signals (NLSs) in the matrix (MA) and NC domains. The nuclear localization of Gag is transient, and export is mediated by a CRM1-dependent NES in the p10 region (6, 52, 53). Thus, it is feasible that Gag may facilitate the nuclear export of the gRNA, either directly or indirectly, to promote particle assembly (53).In support of this idea, Gag mutants engineered to be more efficiently directed to the plasma membrane than wild-type Gag by the addition of the Src membrane-binding domain (in Myr1E virus) or by the insertion of extra basic residues (in SuperM virus) are not concentrated in nuclei when cells are treated with the CRM1 inhibitor leptomycin B (LMB) (8, 20, 53). Moreover, Myr1E and SuperM virus particles incorporate reduced levels of viral gRNA compared to the levels incorporated by wild-type particles. Thus, there is a correlation between the nuclear transit of Gag and gRNA packaging, although the Myr1E and SuperM viruses may be deficient in gRNA encapsidation because they are transported to the plasma membrane too rapidly (8). To test the hypothesis that the loss of Gag nuclear trafficking is responsible for the gRNA packaging defect, we used a gain-of-function genetic approach whereby a heterologous NLS was inserted into Myr1E Gag, yielding mutant virus Myr1E.NLS. Our results revealed that restoring the nuclear trafficking of Myr1E Gag also restored the incorporation of gRNA into mutant virus particles.     

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.     

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.     

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.     

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein.     

Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation

The 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens.