Profiling non-lysyl tRNAs in HIV-1

Abstract

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.

FIGURE 1.

Detection of tRNAs by microarray analysis. (A) Array scheme. Total cellular or viral RNA were deacylated, and directly labeled with a Cy3 or Cy5 containing oligonucleotide using T4 DNA ligase. The labeling samples with the opposite fluorophores were combined and hybridized together on the array. (B) Array specificity. Fluorescence values for the total RNA of HEK293T using human tRNA probes and other probes (Caulobacter tRNA, human rRNA). (C) Array images of one block in the human tRNA microarray hybridized with 293T or HIV-1. Only Cy3 spots are shown for better visualization. The entire array contains 48 blocks, and each probe is repeated 21 times on the array. The particular block shown contains two probes for human tRNA^Lys3, one for tRNA^Asn, and one for a yeast tRNA^Phe standard (SCPhe). Both images have the same contrast for the SCPhe standard. In the array key grid, gray squares indicate probe locations for human tRNAs and open squares indicate probe locations for nonhuman tRNA or rRNA. (D) Selected spots from Cy3-labeled 293T total RNA, wild-type HIV-1, or GagVLP, indicating that significantly lower amounts of tRNA are present in GagVLPs.

FIGURE 2.

Selective packaging of tRNAs into HIV-1. Each bar represents the HIV-1:cell ratio for a specific tRNA. DNA probes on the microarray are complementary to either nuclear-encoded or mitochondrial-encoded tRNAs. (A) Ratios using tRNA in wild-type HIV-1. (B) Ratios using tRNA present in Gag VLPs lacking GagPol. tRNA^Lys1,2 and tRNA^Lys3 probes are indicated by gray arrows, and tRNA^Asn and tRNA^Ile(UAU) probes are indicated by black arrows.

FIGURE 3.

Abundance of tRNAs in HIV-1. tRNA abundance is expressed as percent of total tRNA fluorescence signal in HEK293 tRNA (A) and HIV-1 tRNA (B). Only nuclear-encoded tRNAs are shown. (C,D) Analysis of low molecular weight RNA in HEK293T cells, HIV-1, and Gag VLPs by 2D PAGE. Total RNA was 3′ labeled with ³²pCp. For purposes of quantitative comparison, all samples used equal amounts of viral genomic RNA, were electrophoresed in the same chamber, and exposed to either film or to phosphorimaging at the same time.