5th International Workshop on HIV Drug Resistance & Treatment Strategies


4-8 June 2001, Scottsdale, Arizona


[TITLE:] IMPACT OF AMINO ACID INSERTIONS IN P6 IDENTIFIED IN PATIENTS WITH FAILURE OF COMBINATION ANTIRETROVIRAL THERAPY: ALTERATIONS IN VPR INCORPORATION AND VIRUS MATURATION

[AUTHOR(S):] K Suzuki1, G Kaufmann2, D Jones1, S Piller1, L Leas1, C Harris1, P Cunningham1, P Mallon3, M Mukaide2, A Kelleher1,3 and DA Cooper1,3
1 Centre of Immunology, St Vincent’s Hospital, Sydney, Australia; 2 SRL Tokyo, Japan; and 3 National Centre of HIV Epidemiology and Clinical Research, Sydney, Australia
Antivir Ther. 2001;6 Suppl 1:23

BACKGROUND: We have identified previously undescribed amino acid insertions in HIV p6-gag in three of 450 patients with virological failure after combination antiretroviral (ART). None of these patients had primary mutations in the protease gene. The insertions (4–6 amino acids long) varied slightly in their amino acid sequence but all occurred at a specific location near the C-terminus of p6-gag. Since p6-gag is important for incorporation of Vpr during viral assembly, the effect of these insertions was investigated using recombinant HIV.

METHODS: The p6, protease and reverse transcriptase (RT) genes were amplified from patients’ plasma by RT–PCR and ligated into a genomic HIV-1 clone with pNL4.3 in place of its gag–pol genes. Plasmid DNA was isolated from bacteria transformed with the ligated product. The plasmid DNA was transfected into 293 T cells and recombinant virus was obtained from supernatant. HUT 78 cells were infected with the recombinant virus and RT activity was measured in the cultured supernatant. Western blot (WB) analysis was performed on both cell and virus lysates using an antibody against Vpr.

RESULTS: Recombinant HIV with amino acid insertions in p6-gag region showed grossly altered growth kinetics. Culture in excess of 20 days was required to detect RT activity. Even when detected, RT activity was 200–300 times less than that of wild-type (WT) NL4.3 culture. WB analysis of recombinant virus lysates using an antibody against Vpr indicated lack of full-length vpr incorporation. Sequence analysis of the Vpr gene from patient plasma HIV showed either substituted amino acids in the N-terminal alpha helix (p6-gag binding site) or insertion of an arginine at the C-terminus.

CONCLUSION: We describe novel amino acid insertions in the p6-gag. When these insertions are incorporated into recombinant HIV, the resulting virus is characterized by: (i) extremely low RT activity; and (ii) lack of Vpr incorporation. Moreover, sequence analysis of the vpr gene showed amino acid changes in patients but not in the recombinant virus. These data suggest that the p6 insertions may confer an assembly problem in the recombinant virus, perhaps requiring compensatory amino acid changes within Vpr.

PRESENTING AUTHOR: K Suzuki

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