Lawrence Kleiman

Formation of the tRNALys packaging complex in HIV-1

Human immunodeficiency virus 1 (HIV‐1) uses a host cell tRNALys,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 tRNALys 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 tRNALys 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 tRNALys/LysRS packaging complex hold promise for the development of new anti‐viral agents.

HIV-1 Modulates the tRNA Pool to Improve Translation Efficiency

Abstract 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 tRNALys 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 HIVs 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.

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.

Decreased deoxy-d-glucose transport in Friend cells during exposure to inducers of erythroid differentiation☆

Abstract Various chemicals will induce Friend cells to undergo erythroid differentiation. Concurrent with this differentiation is a large increase in the synthesis of globin messenger RNA (mRNA) and hemoglobin, occurring several days after the initial exposure of the cells to inducer. An earlier change associated with the Friend cell erythroid differentiation is a decrease in the transport of the glucose analogue, 2-deoxy- d -glucose. A substantial transport drop is seen within the first 12 h of exposure to inducer, and this lowered transport rate remains as a characteristic of the induced cell. This change was seen when three different inducers were tested (dimethylsulfoxide, tetramethyl urea and hexamethylene bisacetamide). However, when non-inducible cells (chick embryo and human fibroblasts, and mouse L cells) were exposed to these inducers, no similar decrease in 2-deoxy- d -glucose transport was observed. During induction, this decrease in 2-deoxy- d -glucose transport was associated with a 3-fold drop in the transport Vmax (4.6-1.54 nmoles 2-deoxy- d -glucose/106 cells/min) while no change in the transport Km was observed. l -Glucose uptake was less than 3% of the 2-deoxy- d -glucose uptake at similar hexose concentrations in both induced and uninduced cells. Further, cytochalasin B (CB) (20 μM) inhibited greater than 90% of the deoxy- d -glucose uptake in both cell types. This indicated, most if not all of the total sugar transported was by a carrier-mediated process.

Variable tRNA content in HIV-1IIIB

Low molecular weight RNA in HIV-1 is found in three size classes resembling 7S RNA, 5S RNA, and tRNA. The 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) patterns of tRNA found in HIV-1 have been determined in virus produced in five different cell types: H9, UHC1 (a U937-derived clone), UHC8 (an RT(-) derivative of U937), HeLa, and COS. The presence of the putative primer tRNA for reverse transcriptase, tRNA(Lys,3), has also been determined either by hybridization with a tRNA(Lys,3)-specific DNA probe or by a comparison of the electrophoretic mobility of viral tRNA species with purified human tRNA(Lys,3). Our results indicate the following: 1) The number of tRNA species found in infectious HIV-1IIIB produced in different cell types varies, according to cell type, from greater than 20 to 4, indicating that only 4 or less tRNA species are required for the viral infectious life cycle. 2) There are 1-3 tRNA species tightly associated to the viral genomic RNA, depending upon the cell type producing the virus. 3) The putative primer tRNA, tRNA(Lys,3), is detected with the tRNA(Lys,3)-specific hybridization probe in the tRNA of HIV-1 produced in H9 cells, and the tightly associated tRNA species in this virus has the same electrophoretic mobility in 1-D PAGE as purified tRNA(Lys,3). On the other hand, we cannot detect tRNA(Lys,3) in the tRNA of HIV-1 produced in HeLa cells, and the tightly associated tRNA found in this virus does not migrate with the same electrophoretic mobility as tRNA(Lys,3).

Dual Role for Motif 1 Residues of Human Lysyl-tRNA Synthetase in Dimerization and Packaging into HIV-1

Background: Interaction between lysyl-tRNA synthetase and HIV-1 Gag is critical for tRNALys primer packaging and virus replication. Results: Mutation of residues of dimerization helix 7 abolishes hLysRS packaging into virions, reduces binding to HIV-1 Gag, and affects the synthetase dimerization state and aminoacylation activity. Conclusion: LysRS dimer interface residues interact with HIV-1 Gag. Significance: Mapping host cell-viral protein interaction will aid in development of novel antiviral agents. The primer for reverse transcription in human immunodeficiency virus type 1, human tRNALys,3, is selectively packaged into the virion along with tRNALys1,2. Human lysyl-tRNA synthetase (hLysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also selectively packaged into HIV-1. We have previously defined a tRNALys packaging complex that includes the tRNALys isoacceptors, LysRS, HIV-1 Gag, GagPol, and viral RNA. Numerous studies support the hypothesis that during tRNALys packaging, a Gag·GagPol complex interacts with a tRNALys·LysRS complex, with Gag interacting specifically with the catalytic domain of LysRS, and GagPol interacting with both Gag and tRNALys. 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.

Alterations in the metabolism of transfer RNA during erythroid differentiation of the friend erythroleukemia cell

Abstract In this paper, we report changes in tRNA metabolism we have observed to occur as a result of the tetramethyl urea-induced erythroid differentiation of the Friend erythroleukemia cell. The half-life of tRNA in the uninduced Friend cell in the logarithmic growth phase is 100–122 h, but decreases to 38 h in the induced erythroid cell. The tRNA content per cell is reduced 55%, from 1.76 to 0.793 pg. The calculated rate of accumulation of newly-synthesized tRNA in the Friend cell decreases from 1.62× 10 −3 pg min −1 cell −1 to 2.41 × 10 −4 pg min −1 cell −1 , representing close to a 7-fold reduction in the rate of tRNA accumulation. The decreased half-life of tRNA in the erythroid cell is not due to any gross differences in tRNA post-transcriptional modifications existing between the two cell types. Both the types and proportions of different methylated tRNA nucleosides seem to be identical in both tRNA populations. The major methylated tRNA species found in either cell type are m 1 G, m 2 G, m 2 2 G, m 1 A and m 5 C. Using reverse phase chromatography, changes in tRNA structure can be detected when the uninduced, rapidly-dividing Friend cell enters a non-dividing state either because of high cell density or as a result of erythroid differentiation. Eight of the fifteen tRNA isoacceptor families studied showed changes in their RPC-5 profiles. Five of these families (lysine, arginine, tyrosine, tryptophane, and phenylalanine) show changes in either altered state, while the other three families (alanine, serine, and glycine) are altered only during erythroid differentiation.

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.

Effect of Insulin-Like Growth Factor I on HIV Type 1 Long Terminal Repeat-Driven Chloramphenicol Acetyltransferase Expression

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Isolation and fractionation of retroviral tRNAs

Previous studies concerning the analysis of retroviral tRNA populations involved intracellular metabolic labeling of RNA, followed by the isolation of viral RNA and lengthy sucrose gradient centrifugation for the separation of tRNAs found in various viral compartments. A more rapid, convenient, and safer method for achieving similar aims is described. Isolated total viral RNA is end-labeled in vitro, and tRNA subgroups are fractionated using commercial Nucleobond AX-20 mini columns. 2-D PAGE analysis of mouse mammary tumor virus tRNA fractionated in this way yields gel patterns similar to those obtained with previously described methods.