A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins. Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts. Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA. Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3. We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores. Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs. Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.

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 membranebound 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.

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During human immunodeficiency virus type 1 (HIV-1) assembly, tRNALys isoacceptors are selectively incorporated into virions and tRNA 3 Lys is used as the primer for reverse transcription. We show herein that the tRNA Lys -binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. Lys packaging studies but from tRNA 3 Lys placement studies, which indicate that this protein, and not Pr160 gag-pol , plays a major role in annealing tRNA 3 Lys onto the primer binding site in vitro (9) or in vivo (3).In considering the interactions involved between viral proteins and tRNA Lys during packaging, it must be taken into account that tRNAs have been reported to be channeled from one component of the translational machinery to the next and thus may never be free of this synthetic machinery (26). Such components could involve ribosomes, elongation factors, and aminoacyl-tRNA synthetases. Although it has been shown that elongation factor 1-alpha is packaged into HIV-1 via an interaction with Pr55 gag (5), it is not clear how this protein, which binds to all aminoacylated tRNAs, would confer the ability to selectively package tRNA Lys into the virion. Another tRNAbinding protein in the cytoplasm which is more specific for tRNA Lys is lysyl-tRNA synthetase (LysRS). This enzyme is an attractive candidate for interacting specifically with viral proteins and may play a role in the transport of the three tRNA Lys isoacceptors into the virions. In this work, we will show that during viral assembly, LysRS is in fact nonrandomly packaged into HIV-1 through interaction with Pr55 gag and that a truncated LysRS species associated with selective tRNA Lys packaging is found within the virion. MATERIALS AND METHODSPlasmid construction. SVC21.BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA and was a gift from E. Cohen, University of Montreal. pSVGAG-RRE-R and pSVFS5TprotD25G, which code for Gag and unprocessed Gag-Pol, respectively, have been described previously (24,25). Viral production from either of these two plasmids, which contain the Rev response element (RRE), requires cotransfection with a Rev protein expression vector, such as pCMV-REV. Thus, cotransfection of pSVGAG-RRE-R with pCMV-REV is required to produce virus-like particles containing the unprocessed Pr55 gag precursor protein. In this study, pSVSF5TprotD25G was cotransfected with SVC21P31L, a plasmid coding for HIV-1 proteins including Gag and Rev, but not for stable Gag-Pol. The construction of the mutants SVC21 Dr2 and SVC21 P31L has been described previously (12,19).Cell lines. COS7 cells were maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum and antibiotic. H9, PLB, CEMss, and U937 cell lines were grown in RPMI 1640 with 10% fetal bovine serum and antibiotic.Production of wild-type and mutant HIV-1 virus. Transfection of COS7 cells with the above plasmids by the calcium phosphate method was done as previously described (18). Viruses were isolated from COS7 cell culture mediu...

Cells are categorized as being permissive or nonpermissive according to their ability to produce infectious human immunodeficiency virus type 1 (HIV-1) lacking the viral protein Vif. Nonpermissive cells express the human cytidine deaminase APOBEC3G (hA3G), and Vif has been shown to bind to APOBEC3G and facilitate its degradation. Vif-negative HIV-1 virions produced in nonpermissive cells incorporate hA3G and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that the reduction in DNA production is due to hA3G-facilitated deamination of cytidine, followed by DNA degradation, we provide evidence here that a decrease in the synthesis of the DNA by reverse transcriptase may account for a significant part of this reduction. During the infection of cells with Vif-negative HIV-1 produced from 293T cells transiently expressing hA3G, much of the inhibition of early (>50% reduction) and late (>95% reduction) viral DNA production, and of viral infectivity (>95% reduction), can occur independently of DNA deamination. The inhibition of the production of early minus-sense strong stop DNA is also correlated with a similar inability of tRNA 3 Lys to prime reverse transcription. A similar reduction in tRNA 3 Lys priming and viral infectivity is also seen in the naturally nonpermissive cell H9, albeit at significantly lower levels of hA3G expression.

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription. RNA helicase A (RHA)2 is a member of the DEXH-box (where X can be any amino acid) family of proteins and is also termed DHX9 (1, 2). The DEXH-box proteins, together with the DEAD-box and the Ski2 family members, are referred to as RNA helicases that are able to rearrange the structures of RNA molecules (3). RHA contains a helicase core domain consisting of seven motifs that are conserved for all RNA helicases. Within the N-terminal region of RHA there are two copies of type A double-stranded RNA binding domains. Together with an RGG-box domain located at the C terminus, double-stranded RNA binding domains regulate RNA binding as well as helicase activities of RHA (4). RHA is a nuclear protein and shuttles between the nucleus and the cytoplasm with the assistance of a bidirectional nuclear transport domain consisting of 110 amino acids at the C terminus (5). This function of the RHA nuclear transport domain is subject to regulation of arginine methylation catalyzed by PRMT1 (protein-arginine methyltransferase 1) (6). RHA is able to unwind double-stranded RNA or DNA with the energy derived from hydrolysis of NTPs by virtue of its NTPase activity (1). This property enables RHA to participate in multiple cellular processes from RNA transcription to RNA processing to RNA nuclear export (7). These multiple functions underlie the vital role of RHA in the germ line proliferation and development of Caenorhabditis elegans (8) and also account for the early embryonic lethality observed with RHA knock-out mice (9).The regulation activity of RHA in RNA transcription is implicated by its presence within the RNA polymerase II holoenzyme complex. For example, RHA has been shown to bridge the interactions between RNA polymerase II and transcription co-activators such as CREB-binding protein and BRAC1 (breast cancer-specific tumor suppressor protein 1) (10, 11). RHA also directly interacts with the p65 subunit of NF-B and stimulates NF-B-mediated reporter gene expression (12). Involvement of RHA in transcription is further indicated by the function of its homologue in Drosophila, named the maleless (MLE) gene, that increases gene expression fr...

A 14-amino-acid spacer peptide termed SP1 that separates the capsid (CA) and nucleocapsid (NC) sequences plays an active role in the assembly of human immunodeficiency virus type 1. This activity of SP1 involves its amino-terminal residues that, together with adjacent CA residues, constitute a putative ␣-helical structure spanning Gag residues from positions 359 to 371. In this study, we have determined that the virus assembly determinants within this putative ␣-helix were residues H359, K360, A361, L364, A367, and M368, of which K360 and A367 contribute to virus production to lesser extents. Notably, changes of the two basic amino acids H359 and K360 to arginine (R) impaired virus production, whereas mutations L364I and M368I, in contrast to L364A and M368A, generated near-wild-type levels of virus particles. This suggests that within Gag complexes, amino acids H359 and K360 are involved in stricter steric interactions than L364 and M368. Since L364 and M368 are separated by four residues and thus presumably located on the same side of the helical surface, they may initiate synergistic hydrophobic interactions to stabilize Gag association. Further analysis in the context of the protease-negative mutation D185H confirmed the key roles of amino acids H359, A361, L364, and M368 in virus assembly. Importantly, when transfected cells were subjected to Dounce homogenization and the cell lysates were treated by ultracentrifugation at 100,000 ؋ g, Gag molecules containing each of the H359A, A361V, L364A, and M368A mutations were found mainly in the supernatant fraction (S100), whereas approximately 80% of wild-type Gag proteins were found in the pellet. Therefore, these four mutations must have prevented Gag from generating large complexes.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA 3Lys were additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in vif mutant virions. Moreover, purified vif mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.