3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:53 (abstract no. 4)

Caliendo A, Savara A, An D, Kaplan J, Daeaquila R
Massachusetts General Hospital,MA.

We studied whether mutant RT D67N, K70R, T215F, K219Q HIV-1 replicated better than wild-type in the absence of ZDV because we had previously observed that such a mutant recombinant-expressed RT enzyme was more processive than wild type RT even at the low [dNTP] found in unstimulated PBMC. Equal inocula (3000 TCID₅₀ per 5 million PBMC) of wild-type and mutant virus stocks (from pNL 4-3 background molecular clones) were used to infect either PHA-stimulated, or unstimulated, PBMC from the same blood donor. Unstimulated PBMC were maintained without IL-2 or cell feeds until day 10; PHA and IL-2 were added with medium feeds on days 10, 14, 17, 21, 24. No allogeneic cells were added to initially unstimulated cells. Replication was measured by supernatant p24 antigen and did not differ between mutant and wild-type virus in infections of PHA-stimulated PBMC. In contrast, mutant virus generated significantly higher levels of supernatant p24 antigen than wt in cells stimulated by PHA/IL-2 on day 10 after infection. Wild-type supernatant p24 antigen levels were diminished in cells stimulated after infection compared to cells stimulated before infection. In contrast, mutant virus replicated equally well in cells stimulated either before or after infection. There was a delay of a few days in appearance of fully reverse-transcribed DNA in unstimulated cultures. The observed replication advantage of mutant virus in cells stimulated after infection was not explained by differences in extent of proliferation of cells infected with either wild-type or mutant viruses. A similar in vitro replication advantage over wild-type virus was also seen only in cells stimulated after infection with another AZT-resistant mutant virus containing RT M41L, T215Y, and K219Q. ZDV IC₅₀s for wild-type virus were higher in cells stimulated after infection than in cells stimulated before infection; the potential contribution of this factor to the slow emergence of ZDV resistant mutants in vivo is being modelled. Correlations between in vitro processivity and fidelity of the RT M41L, T215Y, and K219Q mutant enzyme are also underway.

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Copyright © 1996 - Foundation for Retrovirology and Human Health . Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health.