Ongoing replication of wild-type HIV-1 despite selective pressure of 3TC and concurrent replication of drug-resistant viruses in vitro

Kristina Allers, Karin J. Metzner. Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg (Germany).

Background: The early establishment of persistent infection despite highly active antiretroviral therapy (HAART) and immune responses is one of the major obstacles facing the eradication of human immunodeficiency virus type-1 (HIV-1). To further our understanding of the mechanisms of viral persistence, we established an in vitro model to quantify and track the presence of minor populations of wild-type and drug-resistant HIV-1 variants. Material and Methods: Different ratios of wild-type and 3TC-resistant HIV-1 variants (carrying either the M184V or the M184I mutation) were used to infect 174xCEM cells in the presence or absence of 3TC. Supernatants were monitored daily during a follow-up of several weeks. Analyses were carried out using a novel selective and quantitative real time-PCR assay allowing simultaneous quantification of wild-type and drug-resistant HIV-1 variants in the same sample. The assay has a discriminative power of approximately 0.1 percent. Results: Our results show that the persistence of wild-type HIV-1 in the presence of 3TC is dependent on both the 3TC concentration and the initial molar ratios of wild-type and 3TC-resistant virus. Ongoing residual replication of wild-type HIV-1 was observed using an 50% inhibitory concentration of 3TC and was independent of initial molar ratios of input virus. However, using <50% of wild-type HIV-1 for the initial infection, and 3TC in a 95% inhibitory concentration (IC95), the proportion of wild-type viruses decreased below 0.1% within approximately 12 days. Infections with a mixture containing 90% wild-type virus and using an IC95 of 3TC did not result in the disappearance of the wild-type population within 23 days of follow-up. Competition experiments without 3TC revealed that the proportion of drug-resistant variants showed no significant changes or only a slow decline. Conclusion: These results demonstrate that both wild-type and drug-resistant HIV-1 variants are able to persist and replicate, despite culture conditions that may favor eradication.