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.

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.

Dominant role of the 5' TAR bulge in dimerization of HIV-1 genomic RNA, but no evidence of TAR-TAR kissing during in vivo virus assembly.

The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The ΔUCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than ΔUCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.

The contribution of the primer activation signal to differences between Gag- and NCp7-facilitated tRNA(Lys3) annealing in HIV-1.

During tRNA(Lys3) annealing in HIV-1, tRNA(Lys3) binds to both the primer binding site (PBS) and to an 8 nucleotide base-paired sequence upstream of the PBS known as the primer activation signal (PAS). In protease-negative (Pr(-)) HIV-1, the amount of tRNA(Lys3) annealed by Gag is 35% less than that annealed by mature nucleocapsid (NCp7) in protease-positive (Pr(+)) virions. Gag-annealed tRNA(Lys3) also has a reduced ability to initiate reverse transcription, and binds less tightly to viral RNA than NCp7-annealed tRNA(Lys3). Pr(-) virions containing a constitutively single-stranded PAS (2R mutant), show a significant increase in the ability to initiate reverse transcription with little change in the amount of tRNA(Lys3) annealed. However, the 2R mutant does not achieve levels of RT initiation achieved in Pr(+) virions, and tRNA(Lys3) binding to viral RNA remains weak. Wild type levels of initiation and tRNA(Lys3) binding to viral RNA can only be recovered by transient exposure of Pr(-) or Pr(-)2R viral RNA to NCp7. This suggests that in addition to facilitating annealing of tRNA(Lys3) to the PBS and possible denaturation of the PAS, other functions of NCp7 involved in annealing are required. The effect of an inactive protease and/or the 2R mutation upon tRNA(Lys3) annealing and initiation are also observed when the tRNA(Lys3) is annealed in vitro to wild type or mutant viral RNA using either NCp7 or GagDeltap6, indicating a direct effect of the 2R mutation upon tRNA(Lys3) annealing.

Roles of Gag and NCp7 in facilitating tRNA(Lys)(3) Annealing to viral RNA in human immunodeficiency virus type 1.

In protease-negative human immunodeficiency virus type 1 (HIV-1) [Pr(-)], the amount of tRNA(3)(Lys) annealed by Gag is modestly reduced ( approximately 25%) compared to that annealed by mature nucleocapsid (NCp7) in protease-positive HIV-1 [Pr(+)]. However, the tRNA(3)(Lys) annealed by Gag also has a strongly reduced ability to initiate reverse transcription and binds less tightly to viral RNA. Both in vivo and in vitro, APOBEC3G (A3G) inhibits tRNA(3)(Lys) annealing facilitated by NCp7 but not annealing facilitated by Gag. While transient exposure of Pr(-) viral RNA to NCp7 in vitro returns the quality and quantity of tRNA(3)(Lys) annealing to Pr(+) levels, the presence of A3G both prevents this rescue and creates a further reduction in tRNA(3)(Lys) annealing. Since A3G inhibition of NCp7-facilitated tRNA(3)(Lys) annealing in vitro requires the presence of A3G during the annealing process, these results suggest that in Pr(+) viruses NCp7 can displace Gag-annealed tRNA(3)(Lys) and re-anneal it to viral RNA, the re-annealing step being subject to A3G inhibition. This supports the possibility that the initial annealing of tRNA(3)(Lys) in wild-type, Pr(+) virus may be by Gag and not by NCp7, perhaps offering the advantage of Gag's preference for binding to RNA stem-loops in the 5' region of viral RNA near the tRNA(3)(Lys) annealing region.

Interactions of reverse transcriptase sequences in Pol with Gag and LysRS in the HIV-1 tRNALys3 packaging/annealing complex.

During HIV-1 assembly, tRNA(Lys3), the primer for reverse transcriptase (RT) in HIV-1, is selectively packaged into the virus due to a specific interaction between Gag and lysyl-tRNA synthetase (LysRS). However, while Gag alone will incorporate LysRS, tRNA(Lys3) packaging also requires the presence of RT thumb domain sequences in GagPol. The formation of a tRNA(Lys3) packaging/annealing complex involves an interaction between Gag/GagPol/viral RNA and LysRS/tRNA(Lys), and herein, we have investigated whether the transfer of tRNA(Lys3) from LysRS to RT sequences in Pol by a currently unknown mechanism is facilitated by an interaction between LysRS and Pol. We demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. However, cytoplasmic Gag/Pol interactions, detected by either coimmunoprecipitation or incorporation of Pol into Gag viral-like particles, were found to be insensitive to the overexpression or underexpression of LysRS, indicating that a Gag/LysRS/RT interaction is not essential for Gag/Pol interactions. Based on this and previous work, including the observation that the RT connection domain is not required for tRNA(Lys3) packaging, but is required for tRNA(Lys3) annealing, a model is proposed for a tRNA(Lys3) packaging/annealing complex in which the interaction of Gag with Pol sequences during early viral assembly facilitates the retention in budding viruses of both tRNA(Lys3) and early Pol processing intermediates, with tRNA(Lys3) annealing to viral RNA further facilitated by the LysRS/RT interaction.

Function analysis of sequences in human APOBEC3G involved in Vif-mediated degradation.

Human APOBEC3G (hA3G) has been identified as an anti-HIV cellular factor. As a counter measure, the HIV-1 protein Vif causes the degradation of hA3G by binding to it and directing it to the cellular proteasome. In this work, we have used hA3G deletion mutants to map the region in hA3G required for its degradation by Vif to hA3G amino acids 105-245, the linker region between the two zinc coordination motifs. A small fragment of hA3G containing only amino acids 105-245 will undergo Vif-induced degradation. However, while amino acids 105-156 of hA3G are required for Vif interaction with hA3G, they are not themselves sufficient for hA3G degradation, a process that further requires amino acids 157-245. While expression of hA3G fragments 1-156 or 157-384 (but not 246-384) can dominantly inhibit the Vif-mediated degradation of full-length hA3G, only the N-terminal fragment inhibits the Vif/hA3G interaction. Inhibition of hA3G degradation by the C-terminal hA3G fragment 157-384 appears to be related to its ability to prevent the polyubiquitination of hA3G induced by Vif, a process that is required for Vif-mediated proteosomal degradation of hA3G. Non-permissive cells stably expressing hA3G 1-156 or hA3G 157-384 are able to inhibit the replication of wild-type HIV-1, thereby verifying the inhibitory effect of these fragments upon Vif-mediated hA3G degradation and suggesting their potential in anti-HIV-1 therapy.

Inhibition of tRNA3(Lys)-primed reverse transcription by human APOBEC3G during human immunodeficiency virus type 1 replication.

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 (> or =50% reduction) and late (> or =95% reduction) viral DNA production, and of viral infectivity (> or =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.

Cellular distribution of Lysyl-tRNA synthetase and its interaction with Gag during human immunodeficiency virus type 1 assembly.

Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA(3)(Lys), the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i). in the nucleus, (ii). in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii). in mitochondria, and (iv). associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [(35)S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.

The interaction between HIV-1 Gag and APOBEC3G.

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 membrane-bound 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.

Incorporation of pol into human immunodeficiency virus type 1 Gag virus-like particles occurs independently of the upstream Gag domain in Gag-pol.

By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNA(Lys) into Gag VLPs, and primer tRNA(3)(Lys) is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA(3)(Lys), which may be required for Pol and tRNA(3)(Lys) to be retained in the assembling virion until budding is completed.

The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly.

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag.LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208-259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys.