High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo SIV RT mutations leading to a higher fidelity of replication will have a negative impact on viral fitness.
Text by Sarah Lloyd
HIV makes many mistakes during replication due to its error-prone reverse transcriptase (RT) enzyme, facilitating resistance to both anti-HIV drugs and immune responses. Resistance against anti-HIV drugs that target error-prone RT can occur through mutations that result in increased enzyme “fidelity”. We produced a range of RT mutants in the simian immunodeficiency virus model of HIV to test the fitness and fidelity of the mutants in both cell lines and the pigtailed macaque model. The RT mutant viruses were less fit than wild type in cell lines and in macaques. Indeed, the mutants were out-grown by wildtype (WT) in cells and reverted rapidly in animals. We then developed a novel method to study fidelity in infected cells using high-throughput sequencing and showed that a common tenofovir-resistant mutant had a higher fidelity than WT. Understanding high fidelity mutants could open new avenues for addressing HIV escape in drug and vaccine production.
The study came about when we reasoned that HIV (and its monkey counterpart SIV) is so efficient at establishing a chronic infection due to its ability to mutate. If we could impair this ability, it might be more amenable to immune control. Certain drug resistant HIV and SIV variants appeared to have a reduced ability to incorporate errors in biochemical studies, but this hadn’t been studied in replicating viruses or in vivo. Actually, it was difficult to estimate mutation rates of viruses in vitro, requiring deep sequencing, new algorithms, extensive controls and a collaboration with a biostatistics group. We were, however, somewhat impeded with the in vivo studies, as the high fidelity versions either didn’t grow at all, or, if they did, they rapidly reverted to wild type – i.e. the virus’s fidelity wasn’t so high that it couldn’t revert! Nonetheless, our data and techniques exploring RT fidelity in replicating viruses provide formal proof of the high fidelity of drug-resistant RT mutants such as the K65R tenofovir-resistant mutant. With the use of tenofovir becoming more widespread with the advent of pre-exposure prophylaxis (PrEP) in high-risk subjects, understanding the impact of such mutants is important. The rapid reversion we saw of the K65R mutant virus (within 2 weeks) suggests that tenofovir-resistant viruses may be more widespread than believed, since it might well be gone by the time infection is diagnosed (while still being archived in the latent reservoir) – this provides a cautionary note to the use of tenofovir to prevent HIV.
To study mutation rates and reduce experimental error, we developed a novel assay where target cells were co-infected with both wild-type SIV and SIV containing an important drug resistance mutation – K65R. After a single round of replication, we performed high-throughput sequencing around the K65R site. We found significantly fewer mistakes occur during replication of K65R variant, showing it has a higher “fidelity” during virus replication.
Introducing the authors
Pictured: Sarah Lloyd and Stephen Kent.
About the research
High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo
Sarah B. Lloyd, Marit Lichtfuss, Thakshila H. Amarasena, Sheilajen Alcantara, Robert De Rose, Gilda Tachedjian, Hamid Alinejad-Rokny, Vanessa Venturi, Miles P. Davenport, Wendy R. Winnall, Stephen J. Kent
Virology, Volume 492, May 2016, Pages 1–10