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

Saadatmand, Jenan; Guo, Fei; Cen, Shan; Niu, Meijuan; Kleiman, Lawrence

doi:10.1016/j.virol.2008.07.015

Cited by 24 publications

(25 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The simplified model in Figure 6 suggests that binding of hLysRS to the TLE may promote tRNA Lys3 annealing through release of tRNA Lys3 from hLysRS; however, the annealing of tRNA Lys3 to viral RNA takes place within a Gag/GagPol nucleoprotein complex that also promotes this process (Saadatmand and Kleiman 2012). Within this complex, hLysRS has been reported to have multiple interactions with sequences within both Gag Kovaleski et al 2007) and Pol (Saadatmand et al 2008), and we report here an additional interaction with the TLE in the viral RNA. While this interaction may promote tRNA Lys3 annealing to the nearby PBS sequence by facilitating the release of tRNA Lys3 from LysRS, it might also help promote the localization of the hLysRS/tRNA Lys3 -containing nucleoprotein complex to the site of annealing within the large vRNA genome.…”

Section: Discussionmentioning

confidence: 99%

“…The selective incorporation of tRNA Lys isoacceptors appears to be due to a specific interaction between Gag and hLysRS Kovaleski et al 2007). GagPol is also required for binding to tRNA Lys and facilitating its incorporation (Khorchid et al 2000;Kobbi et al 2011), and an interaction between hLysRS and Pol has been proposed (Saadatmand et al 2008;Kobbi et al 2011).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Jones

Saadatmand

Kleiman

et al. 2012

RNA

Self Cite

View full text Add to dashboard Cite

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 primerbinding 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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Jones

Saadatmand

Kleiman

et al. 2012

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…Catalytically inactive RT or alternative RT structural forms may have produced the observed result. Specifically, the packaging and annealing of tRNA into virions involve a complex set of interactions between the tRNA, lysyl-tRNA synthetase, and Gag and Gag/Pol polyproteins (7,32). It is known that tRNA 3 Lys can stimulate polymerization by p66 homodimers, induce conformational changes, and increase proteolytic cleavage of the RNase H domain (3).…”

Section: Discussionmentioning

confidence: 99%

“…Purified tRNA 3 Lys from human placenta (Bio S&T, Canada) was 5Ј end labeled by [␥- 32 P]ATP as previously described (43) and was annealed to D199 at a primer/template (P/T) ratio of 1:2. After binding with purified RT, the reactions were initiated by adding 6 mM MgCl 2 and 500 M dNTPs in a reaction buffer containing 25 mM Tris-HCl, pH 8.0, 25 mM NaCl, and 0.5 mM EDTA and DTT.…”

mentioning

confidence: 99%

Reduced Fitness in Cell Culture of HIV-1 with Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Mutations Correlates with Relative Levels of Reverse Transcriptase Content and RNase H Activity in Virions

Wang

Bambara

Demeter

et al. 2010

J Virol

View full text Add to dashboard Cite

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of multidrug therapy for HIV-1. Understanding the effect of NNRTI-resistant mutants on virus replication and reverse transcriptase (RT) function is valuable for the development of extended-spectrum NNRTIs. We measured the fitness of six NNRTI-resistant mutants, the K103N, V106A, Y181C, G190A, G190S, and P236L viruses, using a flow cytometry-based cell culture assay. K103N and Y181C viruses had fitness similar to that of the wild type while V106A, G190A, G190S, and P236L viruses had reduced fitness. We also determined the biochemical correlates of fitness by measuring the RNase H and polymerization activities of recombinant mutant RTs and virion-associated RTs. The RNase H activities of recombinant and virion-associated RTs correlated with the relative fitness for each mutant. K103N and Y181C mutants had normal RNase H activity; V106A, G190A, and G190S mutants had moderate reductions in activity; and the P236L mutant had substantially reduced activity. With the exception of the P236L mutant, reduced fitness correlates with low virion-associated polymerization efficiency and reduced RT content. Reduced polymerase function in virions derived from low RT content rather than an intrinsic polymerization defect in each RT protein. In conclusion, severe defects in RNase H activity alone, exemplified by the P236L mutant, appear sufficient to cause a substantial reduction in fitness. For the other NNRTI mutants, reductions in RT content decreased both polymerization and RNase H activity in virions. RNase H reduction was compounded by intrinsic RNase H defects in the mutant RTs.HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that converts the single-stranded RNA viral genome into double-stranded DNA. It has RNA-and DNA-dependent DNA polymerase activities, which copy RNA or DNA templates, respectively, into double-stranded DNA, as well as RNase H activity that cleaves genomic RNA still hybridized to DNA (40). Although both activities are essential for HIV-1 replication, some studies have shown that polymerization and RNase H activities are not rigorously coupled (11,12,27,36). RT is a heterodimer comprised of two subunits, a 66-kDa (p66) subunit and a 51-kDa (p51) subunit (20). The p66 subunit has both the polymerase and RNase H domains, which make contact simultaneously with the RNA genome (12). The p51 subunit is an N-terminal cleavage product of p66 which lacks the RNase H domain and serves as a structural scaffold for the p66 subunit (2).Because of its essential role in the virus life cycle, RT is an important antiretroviral target for the treatment of HIV-1. Nonnucleoside RT inhibitors (NNRTIs) are one of three classes of antiretroviral RT inhibitors that are commonly used in treatment. They are a group of structurally diverse small hydrophobic compounds, which can specifically bind and inhibit RT (reviewed in reference 34). One limitation of NNRTIs is that they face a low genetic barrier to resistance. Previous studies o...

show abstract

“…Findings from coimmunoprecipitation assays and Western blot analyses suggest that HIV-1 RT interacts with Gag for RT packaging (347,348). HIV-1 matrix Gag and p6 Gag in Gag might interact with RT, covering the thumb domain of RT (347).…”

Section: Gag-rt Interactionmentioning

confidence: 99%

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Clercq

2016

Microbiol Mol Biol Rev

View full text Add to dashboard Cite

SUMMARYThe HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle.

show abstract

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

Cited by 24 publications

References 45 publications

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

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

Reduced Fitness in Cell Culture of HIV-1 with Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Mutations Correlates with Relative Levels of Reverse Transcriptase Content and RNase H Activity in Virions

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Contact Info

Product

Resources

About

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

Cited by 24 publications

References 45 publications

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

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

Reduced Fitness in Cell Culture of HIV-1 with Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Mutations Correlates with Relative Levels of Reverse Transcriptase Content and RNase H Activity in Virions

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Contact Info

Product

Resources

About

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

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