Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells.

In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5' leader and 3' trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5' leader and long 3' trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.

Inhibition of HIV-1 assembly by coiled-coil domain containing protein 8 in human cells.

Human Immunodeficiency Virus type 1 (HIV-1) major structure protein Gag is synthesized in the cytoplasm, assembles on the plasma membrane, subsequently buds and releases. HIV-1 viral particles incorporate a number of host proteins to facilitate or inhibit HIV-1 replication. Here we identify a new host protein, coiled-coil domain containing protein 8 (CCDC8), in HIV-1 particles. Incorporation of CCDC8 into virions is dependent on the interaction between CCDC8 and Gag matrix region. Exogenous overexpression of CCDC8 can strongly inhibit HIV-1 production, up to ~30 fold. CCDC8 is a membrane-associated protein. The interaction between exogenously expressed CCDC8 and Gag on the plasma membrane changes the assembly of Gag, and redirects it into intracellular sites, or causes Gag endocytosis. CCDC8, along with cytoskeleton protein obscuring-like1 (Obsl1) and E3 ligase Cul7, induces Gag polyubiquitination and degradation. Thus we identify a new host protein and a new pathway for HIV-1 Gag polyubiquitination and degradation. This pathway presents potential therapeutic strategies against HIV infection.

In virio SHAPE analysis of tRNA(Lys3) annealing to HIV-1 genomic RNA in wild type and protease-deficient virus.

BACKGROUND: tRNA(Lys3) annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNA(Lys3) annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNA(Lys3) to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNA(Lys3), resulting in a more tightly bound and efficient primer for reverse transcription. RESULTS: In this report, we have used in virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNA(Lys3) to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNA(Lys3) annealed by Gag in Pr- virions reflects both missing interactions of tRNA(Lys3) with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA. CONCLUSIONS: We propose secondary structure models for the tRNA(Lys3)/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNA(Lys3) to HIV-1 viral RNA, followed by a more complete annealing by NCp7.

Role of the OB-fold of RNA helicase A in the synthesis of HIV-1 RNA.

RNA helicase A (RHA), a DExD/H protein, contains a stretch of repeated arginine and glycine-glycine (RGG) residues and an oligonucleotide/oligosaccharide-binding fold (OB-fold) at the C-terminus. RHA has been reported to function as a transcriptional cofactor. This study shows the role of RGG and OB-fold domains of RHA in the activation of transcription and splicing of HIV-1 RNA. RHA stimulates HIV-1 transcription by enhancing the occupancy of RNA polymerase II on the proviral DNA. Deletion of RGG or both RGG and OB-fold does not change the transcriptional activity of RHA, nor does the stability of viral RNA. However, deletion of both RGG and OB-fold rather than deletion of RGG only results in less production of multiply spliced 6D RNAs. The results suggest that the OB-fold is involved in modulating HIV-1 RNA splicing in the context of some HIV-1 strains while it is dispensable for the activation of HIV-1 transcription.

The role of A-kinase anchoring protein 95-like protein in annealing of tRNALys3 to HIV-1 RNA.

BACKGROUND: RNA helicase A (RHA), a DExH box protein, promotes annealing of tRNALys3, a primer for reverse transcription, to HIV-1 RNA and assembles into virus particles. A-kinase anchoring protein 95-like protein (HAP95) is a binding partner of RHA. The role of HAP95 in the annealing of tRNALys3 was examined in this study. RESULTS: HAP95 associates with the reverse transcriptase region of Pol protein of HIV-1. Decreasing endogenous HAP95 in HIV-1-producing 293T cells by siRNA reduces the amount of tRNALys3 annealed on viral RNA. This defect was further deteriorated by knockdown of RHA in the same cells, suggesting a cooperative effect between these two proteins. Biochemical assay in vitro using purified GST-tagged HAP95 shows that HAP95 may inhibit the activity of RHA. CONCLUSION: The results support a hypothesis that HAP95 may transiently block RHA's activity to protect the annealed tRNALys3 on viral RNA in the cells from removing by RHA during the packaging of RHA into virus particles, thus facilitating the annealing of tRNALys3 to HIV-1 RNA.

Helicase associated 2 domain is essential for helicase activity of RNA helicase A.

RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine-glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity.

Human APOBEC3F incorporation into human immunodeficiency virus type 1 particles.

APOBEC3 proteins are a family of cytidine deaminases that exhibit broad antiretroviral activity. Among APOBEC3 proteins, APOBEC3G (hA3G) and APOBEC3F (hA3F) exhibit the most potent anti-HIV-1 activities. Although the incorporation of hA3F into virions is a prerequisite for exerting its antiviral function, the detail mechanism underlying remains incompletely understood. In this work, we present data showing that the nucleocapsid (NC) domain of HIV-1 Gag and a linker sequence between the two cytidine deaminase domains within hA3F, i.e., 104-156 amino acids, are required for viral packaging of hA3F. A detailed mapping study reveals that the cluster of basic residues surrounding the N-terminal zinc finger (ZF) and the linker region between the ZFs of HIV-1 NC play an important role in A3F incorporation, in addition, at least one of two ZFs is required. A hA3F fragment is able to compete with both hA3G and hA3F for viral incorporation, suggesting a common mechanism underlying virion encapsidation of hA3G and hA3F. Taken together, these results shed a light on the detail mechanism underlying viral incorporation of hA3F.

Repetitive RNA unwinding by RNA helicase A facilitates RNA annealing.

Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding of helicases contributes to their biological functions.

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell.

BACKGROUND: RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells. METHODS: The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A-RNA interaction and RNA helicase A-stimulated viral RNA processes. RESULTS: Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA(Lys3) annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A-HIV-1 RNA interaction in the cells. CONCLUSIONS: The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A. GENERAL SIGNIFICANCE: The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.

Roles of the linker region of RNA helicase A in HIV-1 RNA metabolism.

RNA helicase A (RHA) promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNA(Lys3) to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs) that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNA(Lys3) annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNA(Lys3) annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5'-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging.

The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

Dual role for motif 1 residues of human lysyl-tRNA synthetase in dimerization and packaging into HIV-1.

The primer for reverse transcription in human immunodeficiency virus type 1, human tRNA(Lys,3), is selectively packaged into the virion along with tRNA(Lys1,2). Human lysyl-tRNA synthetase (hLysRS), the only cellular factor known to interact specifically with all three tRNA(Lys) isoacceptors, is also selectively packaged into HIV-1. We have previously defined a tRNA(Lys) packaging complex that includes the tRNA(Lys) isoacceptors, LysRS, HIV-1 Gag, GagPol, and viral RNA. Numerous studies support the hypothesis that during tRNA(Lys) packaging, a Gag·GagPol complex interacts with a tRNA(Lys)·LysRS complex, with Gag interacting specifically with the catalytic domain of LysRS, and GagPol interacting with both Gag and tRNA(Lys). In this work, we have identified residues along one face of the motif 1 dimerization helix (H7) of hLysRS that are critical for packaging of the synthetase into virions. Mutation of these residues affects binding to Gag in vitro, as well as the oligomerization state and aminoacylation activity of the synthetase. Taken together, these data suggest that H7 of LysRS has a dual function. In its canonical role it maintains the synthetase dimer interface, whereas in its function in tRNA primer recruitment, it bridges interactions with HIV-1 Gag.

In vitro and in vivo analysis of the interaction between RNA helicase A and HIV-1 RNA.

RNA helicase A (RHA) promotes multiple steps of HIV-1 RNA metabolism during viral replication, including transcription, translation, and the annealing of primer tRNA(3)(Lys) to the viral RNA. RHA is a member of the DExH subclass of RNA helicases that uniquely contains two double-stranded RNA binding domains (dsRBDs) at its N terminus. Here, we performed a genome-wide analysis of the interaction of RHA with HIV-1 RNA both in vitro, using fluorescence polarization, and during viral replication, using an RNA-protein coprecipitation assay. In vitro, RHA binds to all the isolated regions of the HIV-1 RNA genome tested, with K(d) (equilibrium dissociation constant) values ranging from 44 to 178 nM. In contrast, during viral replication, RNA-protein coprecipitation assays detected only a major interaction of RHA with the 5'-untranslated region (5'-UTR) and a minor interaction with the Rev response element (RRE) of HIV-1 RNA. Since RHA does not associate well with all the highly structured regions of HIV-1 RNA tested in vivo, the results suggest that other viral or cellular factors not present in vitro may modulate the direct interaction of RHA with HIV-1 RNA during virus replication. Nevertheless, a role for duplex RNA as a target for RHA binding in vivo is suggested by the fact that the deletion of either one or both dsRBDs eliminates the in vivo interaction of RHA with HIV-1 RNA. Furthermore, these mutant RHAs do not promote the in vivo annealing of tRNA(3)(Lys) to viral RNA, nor are they packaged into virions, demonstrating that the dsRBDs are essential for the role of RHA in HIV-1 replication.

Aspects of HIV-1 assembly that promote primer tRNA(Lys3) annealing to viral RNA.

The major cellular tRNA(Lys) isoacceptors are tRNA(Lys1,2) and tRNA(Lys3). During the replication of human immunodeficiency virus 1 (HIV-1), tRNA(Lys3) is used to prime reverse transcription of the viral RNA genome into double-stranded DNA, which is then integrated into the host genome. The annealing of tRNA(Lys3) to 5'-terminal sequences of viral RNA is multi-staged, with an initial poor quality, cytoplasmic annealing promoted by the Gag precursor protein, followed by a more effective annealing imposed upon the Gag-annealed tRNA(Lys3) that occurs after viral protein processing, and that is facilitated by mature nucleocapsid (NCp7). The initial annealing by Gag is assisted by the architecture of an early viral assembly intermediate we term the "tRNA(Lys3) annealing complex" whose composition includes Gag, GagPol, viral RNA, lysyl-tRNA synthetase (LysRS), and the tRNA(Lys) isoacceptors. Our model proposes that the reverse transcriptase sequences in GagPol bind all tRNAs non-specifically, and that the cytoplasmic tRNA population to which GagPol is exposed is enriched in tRNA(Lys) isoacceptors due to a specific interaction between Gag and LysRS. We further predict a protein conformation within the annealing complex that not only promotes this tRNA(Lys) enrichment, but that also facilitates the transfer of tRNA(Lys3) from GagPol to the viral RNA where annealing is carried out by nucleocapsid sequences within Gag.

Cyclic peptide inhibitors of HIV-1 capsid-human lysyl-tRNA synthetase interaction.

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNA(Lys,3), which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents.

HIV-1 modulates the tRNA pool to improve translation efficiency.

Despite its poorly adapted codon usage, HIV-1 replicates and is expressed extremely well in human host cells. HIV-1 has recently been shown to package non-lysyl transfer RNAs (tRNAs) in addition to the tRNA(Lys) needed for priming reverse transcription and integration of the HIV-1 genome. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding codons that are highly used by HIV-1 but avoided by its host are overrepresented in HIV-1 virions. In particular, tRNAs decoding A-ending codons, required for the expression of HIV's A-rich genome, are highly enriched. Because the affinity of Gag-Pol for all tRNAs is nonspecific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Codon usage of HIV-1 early genes is similar to that of highly expressed host genes, but codon usage of HIV-1 late genes was better adapted to the selectively enriched tRNA pool, suggesting that alterations in the tRNA pool are induced late in viral infection. If HIV-1 genes are adapting to an altered tRNA pool, codon adaptation of HIV-1 may be better than previously thought.

Coordinate roles of Gag and RNA helicase A in promoting the annealing of formula to HIV-1 RNA.

RNA helicase A (RHA) has been shown to promote HIV-1 replication at both the translation and reverse transcription stages. A prerequisite step for reverse transcription involves the annealing of tRNA(3)(Lys), the primer for reverse transcription, to HIV-1 RNA. tRNA(3)(Lys) annealing is a multistep process that is initially facilitated by Gag prior to viral protein processing. Herein, we report that RHA promotes this annealing through increasing both the quantity of tRNA(3)(Lys) annealed by Gag and the ability of tRNA(3)(Lys) to prime the initiation of reverse transcription. This improved annealing is the result of an altered viral RNA conformation produced by the coordinate action of Gag and RHA. Since RHA has been reported to promote the translation of unspliced viral RNA to Gag protein, our observations suggest that the conformational change in viral RNA induced by RHA and newly produced Gag may help facilitate the switch in viral RNA from a translational mode to one facilitating tRNA(3)(Lys) annealing.

Formation of the tRNALys packaging complex in HIV-1.

Human immunodeficiency virus 1 (HIV-1) uses a host cell tRNA(Lys,3) molecule to prime reverse transcription of the viral RNA genome into double-stranded DNA prior to integration into the host genome. All three human tRNA(Lys) isoacceptors along with human lysyl-tRNA synthetase (LysRS) are selectively packaged into HIV-1. Packaging of LysRS requires the viral Gag polyprotein and incorporation of tRNA(Lys) additionally requires the Gag-Pol precursor. A model that incorporates the known interactions between components of the putative packaging complex is presented. The molecular interactions that direct assembly of the tRNA(Lys)/LysRS packaging complex hold promise for the development of new anti-viral agents.

Profiling non-lysyl tRNAs in HIV-1.

During its assembly, human HIV-1 selectively packages the tRNA(Lys) isoacceptors, including tRNA(Lys3), the primer for the reverse transcriptase. However, other low molecular weight RNA species are also seen in the virus. We profiled the tRNAs packaged into HIV-1 using microarray analysis and validated our results by two-dimensional gel electrophoresis and RT-PCR. In addition to tRNA(Lys) isoacceptors, tRNA(Asn) and the rare isoacceptor of tRNA(Ile) are also selectively packaged. In Gag viral-like particles missing the GagPol protein, overall tRNA incorporation is reduced by >80%. This reduction is significantly greater than can be accounted for by the reduction in tRNA(Lys) isoacceptors, tRNA(Asn) and tRNA(Ile), suggesting that incorporation of other tRNAs may also require the GagPol protein. These results demonstrate selective incorporation of non-lysyl tRNAs into HIV-1 and highlight the application of microarrays as a novel method to study tRNA incorporation into viruses.