Mutations in the 5′ End of the Human Immunodeficiency Virus Type 1 Polypurine Tract Affect RNase H Cleavage Specificity and Virus Titer

Abstract

The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5 end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5 end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5 end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

Effects of Mutations in the G Tract of the Human Immunodeficiency Virus Type 1 Polypurine Tract on Virus Replication and RNase H Cleavage

“…2. When cells were infected with a virus containing the wild-type PPT, the percentages of consensus and aberrant 2-LTR circle junction sequences were very similar to previously reported results (14,21). Fisher's exact tests were performed to determine if mutations caused statistically significant differences in the 2-LTR circle junction sequences.…”

“…G-to-T mutations at these positions (T-2-5) affected the cleavage specificity in a very defined fashion: the proportion of DNAs containing a 1-bp insertion derived from the PPT increased. It is plausible that when the contacts of the nucleic acid at positions Ϫ2 and Ϫ5 with Q475 and Y501 of RT, We previously characterized the effects of mutations in the 5Ј end of the PPT on virus replication (21). Single and double mutations had small effects on the virus titer; a complex mutation involving seven of the first nine nucleotides from the 5Ј end of the PPT had a large effect on the titer and on the 2-LTR circle junction populations.…”

“…previously described (21). The plasmids were analyzed by restriction endonuclease digestion and DNA sequencing to confirm that (only) the desired mutations were present in the vectors.…”

“…Linear DNAs are substrates for nonhomologous end-joining reactions that generate two-long-terminalrepeat (2-LTR) circles (20). We have used the sequences of 2-LTR circle junctions to show that mutations in the 5Ј end of the PPT alter the specificity of RNase H cleavage in vivo (21). We wanted to determine if mutations in the G tract also affect the specificity of RNase H cleavage in vivo.…”

Mutations in the U5 Sequences Adjacent to the Primer Binding Site Do Not Affect tRNA Cleavage by Rous Sarcoma Virus RNase H but Do Cause Aberrant Integrations In Vivo

“…More specifically, the left (U3) long terminal repeat (LTR) terminus of the linear dsDNA molecule is defined by the removal of PPT primer. In the case of HIV-1, the PPT sequence is an important determinant for the proper generation and removal of the PPT primer by HIV-1 RNase H (7,17,18,23,26). The right (U5) LTR terminus of the linear dsDNA molecule is defined by the removal of the tRNA used to initiate minus-strand DNA synthesis.…”