HIV-1 designed to use different tRNAGln isoacceptors prefers to select tRNAThr for replication

Abstract

Background: Previous studies have shown that infection with human immunodeficiency virus type 1 (HIV-1) causes acceleration of the synthesis of glutamine tRNA (tRNAGln) in infected cells. To investigate whether this might influence HIV-1 to utilize tRNAGln as a primer for initiation of reverse transcription, we have constructed HIV-1 proviral genomes in which the PBS and the A-loop region upstream of the PBS have been made complementary to either the anticodon region of tRNAGln,1 or tRNAGln,3 and 3' terminal 18 nucleotides of each isoacceptor of tRNAGln.

Results: Viruses in which the PBS was altered to be complementary to tRNAGln,1 or tRNAGln,3 with or without the A-loop all exhibited a lower infectivity than the wild type virus. Viruses with only the PBS complementary to tRNAGln,1 or tRNAGln,3 reverted to wild type following culture in SupT1 cells. Surprisingly, viruses in which the PBS and A-loop were complementary to tRNAGln,1 did not grow in SupT1 cells, while viruses in which the PBS and A-loop were made complementary to tRNAGln,3 grew slowly in SupT1 cells. Analysis of the PBS of this virus revealed that it had reverted to select tRNAThr as the primer, which shares complementarity in 15 of 18 nucleotides with the PBS complementary to tRNAGln,3.

Conclusion: The results of these studies support the concept that the HIV-1 has preferred tRNAs that can be selected as primers for replication.

Figure 1

tRNA^Gln and mutated proviral genomes. Panel A. Cloverleaf structure of tRNA^Gln,1 and tRNA^Gln,3. tRNA^Gln,1 (major) and tRNA^Gln,3 (minor) are depicted. The tRNAs differ in the nucleotides within the PBS (boxed) as well as nucleotides in the anticodon region (boxed). The modified nucleotides are noted. The structures are taken from Kuchino et al. [22]. Panel B. Modifications in the NL-4 proviral genome. NL-4 WT refers to the wild type NL-4 genome with the PBS and A-loop complementary to tRNA^Lys,3. NL-4-Gln1 refers to a modified proviral genome in which the PBS was modified to be complementary to the 3' terminal nucleotides of tRNA^Gln,1. NL-4 Gln1-AC also contains a PBS complementary to the 3' terminal nucleotides of tRNA^Gln,1 with additional modifications of the A-loop region (GAGTCAG) noted in bold. NL-4-Gln3 is an HIV-1 with the PBS modified to be complementary to the 3' terminal 18-nucleotides of tRNA^Gln,3. Note that the PBS is nearly identical with the exception of the T to C change in the PBS. NL-4 Gln3-AC refers to an HIV-1 in which the PBS was modified to be complementary to tRNA^Gln,3 with additional modification in the A-loop region consisting of GAGTCAA which is complementary to the anticodon region of tRNA^Gln3.

Figure 2

Replication of HIV with PBS and A-loop complementary to tRNA^Gln. Panel A. Replication of wild type and viruses with PBS complementary to tRNA^Gln,1. Infections were established in SupT1 cells with equal amounts of virus as determined by infectious units. p24 antigen was then assayed in the culture supernatants at weekly intervals following initiation of the experiment. Values for the wild type virus increased to greater than 10⁴ nanograms/ml by approximately 14 days following initiation of the infection. The cultures were terminated at day 28. Viruses derived from NL-4-Gln1 and NL-4-Gln1-AC were carried out to approximately 56 days post initiation of culture. Note that viruses derived from NL-4-Gln1-AC did not grow, as evidenced by p24 antigen that were near the levels of mock infected cells. Cultures were terminated at day 56. Data is representative from three independent experiments. Panel B. Replication of viruses with the PBS complementary to tRNA^Gln,3. The replication of the wild type virus is depicted. Cultures initiated with viruses derived from NL-4-Gln3 and NL-4-Gln3-AC were monitored over 56 days of culture. The viruses derived from NL-4-Gln3 eventually reached levels approximating that of the wild type virus by day 42 through 56. Viruses derived from NL-4-Gln3-AC demonstrated a slow and gradual increase reaching levels approximately 1/100 of that of the wild type virus at the time of termination of the culture (day 56). Data is representative of three independent experiments.

Figure 3

Sequence complementarity of tRNA^Gln and tRNA^Thr with mutant proviral genomes. Panel A. Sequence complementary of tRNA^Gln,3 with NL-4-Gln3-AC. Depicted is the predicted complementarity between the 3' terminal nucleotides and the PBS and the anticodon of tRNA^Gln,3 with the modified A-loop region of NL-4-Gln3-AC. Panel B. Complementarity between 3' terminal nucleotides of tRNA^Thr with the PBS of NL4-Gln3-AC. Nucleotide differences within the PBS and tRNA^Thr are underlined. The anticodon region of tRNA^Thr has complementarity with the modified A-loop region of NL-4-Gln3. Additional complementarity between tRNA^Thr and the PBS of NL-4-Gln1-AC is also shown. The single nucleotide difference between the PBS is underlined. The resulting GC pair of tRNA^Thr and the PBS of NL-4-Gln3-AC should be compensated for by a GU base pair. Note also the predicted complementarity between the anticodon region of tRNA^Thr with the modified A-loop region of NL-4-Gln1-AC.