Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis

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.

Download Full-text

Mutations in the Primer Grip of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Impair Proviral DNA Synthesis and Virion Maturation

Journal of Virology ◽

10.1128/jvi.72.9.7676-7680.1998 ◽

1998 ◽

Vol 72 (9) ◽

pp. 7676-7680 ◽

Cited By ~ 25

Author(s):

Qiang Yu ◽

Michele Ottmann ◽

Christine Pechoux ◽

Stuart Le Grice ◽

Jean-Luc Darlix

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Dna Synthesis ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Assembly ◽

Conserved Residues ◽

Proviral Dna ◽

Immunodeficiency Virus

ABSTRACT This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the gag-pol precursor, prior to virion budding, indicating that intracellular stability of Pr160 gag-pol is of key importance during virus assembly.

Download Full-text