Biomedical subjects

Lawrence Kleiman

Publications and source records attributed to Lawrence Kleiman.

26 records · Page 2Linked to original sources

PubMed2003

Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1.

During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [(35)S]Cys-[(35)S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.

Animals↗

PubMed2002

Correlation between tRNALys3 aminoacylation and its incorporation into HIV-1.

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) is selectively packaged into the virus, where tRNA(Lys3) serves as the primer for reverse transcription. Lysyl-tRNA synthetase is also selectively incorporated into HIV-1 and is therefore a strong candidate for being the signal by which viral proteins interact with tRNA(Lys) isoacceptors. Previously, mutations in the tRNA(Lys3) anticodon have been shown to strongly inhibit the charging of tRNA(Lys3) by lysyl-tRNA synthetase in vitro, and we show here that in vivo aminoacylation is also inhibited by anticodon changes. The order of decreasing in vivo aminoacylation for tRNA(Lys3) anticodon mutants is: wild-type SUU (where S = mcm(5)S(2)U) 100%) --> SGU (49%) --> CGU (40%) --> SGA (0%) and CGA (0%). We found that the ability of these tRNA(Lys3) anticodon variants to be aminoacylated in vivo is directly correlated with their ability to be packaged into HIV-1. These data showed that the anticodon is a major determinant for tRNA(Lys3) packaging and support the conclusion that its productive interaction with lysyl-tRNA synthetase is important for tRNA(Lys3) incorporation into HIV-1.

Animals↗

PubMed2002

tRNA(Lys3): the primer tRNA for reverse transcription in HIV-1.

During the assembly of HIV-1, tRNA(Lys) isoacceptors are selectively packaged into the virion, and one of these, tRNA(Lys3), is annealed to the viral RNA genome where it acts to prime the reverse transcriptase (RT)-catalyzed synthesis of viral DNA. We review herein what is known about the selective packaging and annealing of primer tRNA(Lys3). Current evidence suggests that a complex of two major precursor viral proteins, Pr55gag and Pr160(gag-pol), interact with a tRNA(Lys)/lysyl-tRNA synthetase (LysRS) complex during viral assembly, with Pr55gag interacting with both LysRS and Pr160(gag-pol), and RT sequences within Pr160(gag-pol) binding to tRNA(Lys). LysRS appears to target the tRNA(Lys) isoacceptors for incorporation into HIV-1. In the virion, the 3' terminal 18 nucleotides of tRNA(Lys3) anneals to an 18-nucleotide sequence at the 5' terminal region of viral RNA termed the primer binding site (PBS). Evidence is presented that other regions on the tRNA(Lys3) also anneal with other regions in viral RNA upstream of the PBS, resulting in a destabilized tRNA(Lys3) structure. Both viral and tRNA(Lys3) regions need to be denatured to establish annealing, and the roles of both viral and cellular proteins in this process are discussed.

Base Sequence↗

PubMed2002

Effect of altering the tRNA(Lys)(3) concentration in human immunodeficiency virus type 1 upon its annealing to viral RNA, GagPol incorporation, and viral infectivity.

Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys)(3) as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNA(Lys), tRNA(Lys)(3). Increasing the cytoplasmic concentration of tRNA(Lys)(3) through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA(Lys)(3) gene results in a greater incorporation of tRNA(Lys)(3) into virions, which is accompanied by increased annealing of tRNA(Lys)(3) to the viral genome and increased infectivity of the viral population. Increased viral tRNA(Lys)(3) is accompanied by decreased viral tRNA(Lys)(3), with the total tRNA(Lys)/virion and the GagPol/Gag ratios remaining unchanged. Viral tRNA(Lys) can be doubled, with increases in both tRNA(Lys)(3) and tRNA(Lys)(1,2) concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA(Lys)(3) annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNA(Lys) incorporation into HIV-1, whereas LysRS is.

Animals↗

PubMed2002

Characterization of a putative alpha-helix across the capsid-SP1 boundary that is critical for the multimerization of human immunodeficiency virus type 1 gag.

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 alpha-helical structure spanning Gag residues from positions 359 to 371. In this study, we have determined that the virus assembly determinants within this putative alpha-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 x 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.

Amino Acid Sequence↗

PubMed2002

Retrovirus-specific packaging of aminoacyl-tRNA synthetases with cognate primer tRNAs.

The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are, tRNA(Trp), and tRNA(Pro), respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNA(Pro) in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.

3T3 Cells↗

PubMed2002

The Tat protein of human immunodeficiency virus type 1 (HIV-1) can promote placement of tRNA primer onto viral RNA and suppress later DNA polymerization in HIV-1 reverse transcription.

Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.

DNA Primers↗

PubMed2002

Role of RNA in facilitating Gag/Gag-Pol interaction.

We have examined the influence of RNA upon the interaction of Gag-Pol with Gag during human immunodeficiency virus type 1 (HIV-1) assembly. COS7 cells were transfected with protease-negative HIV-1 proviral DNA, and Gag/Gag-Pol complexes were detected by coimmunoprecipitation with anti-integrase. In COS7 cells, Gag/Gag-Pol is found almost entirely in pelletable, membrane-bound complexes. Exposure of cells to 1% Triton X-100 releases Gag/Gag-Pol from bulk membrane, but the complexes remain pelletable. The role of RNA in facilitating the interaction between Gag and Gag-Pol was examined in these bulk membrane-free, pelletable complexes. The specific presence of viral genomic RNA is not required to maintain the Gag/Gag-Pol interaction, but some type of RNA is, since exposure to RNase destabilized the Gag/Gag-Pol complex. When present only in Gag, the nucleocapsid mutation R7R10K11S, which inhibits Gag binding to RNA, inhibits the formation of both Gag and Gag/Gag-Pol complexes. When present only in Gag-Pol, this mutation has no effect upon complex formation. This result indicates that Gag-Pol may not interact directly with RNA but rather requires RNA-facilitated Gag multimerization for its interaction with Gag.

Animals↗