Background
People living with HIV (PLWH) receiving HIV integrase (IN) strand transfer inhibitors (INSTIs) have been reported to experience virological failure (VF) in the absence of resistance mutations in IN. To elucidate INSTI resistance mechanisms and pathways, we performed long-term (~ one year) passaging of several viral isolates with an escalating concentration of the INSTI dolutegravir (DTG). Independent of viral strain and cell type, HIV-1 became resistant to DTG by sequentially acquiring mutations in Env, nucleocapsid (NC), and, in some cases, IN. This study aims to elucidate the role of NC in HIV-1 resistance to INSTIs.
Methods
We evaluated the impacts of the selected NC mutations on DTG sensitivity and viral infectivity. qPCR and time-of-addition (TOA) assays were conducted to examine the impacts of mutations on post-entry events in the virus replication cycle.
Results
The NC mutations selected in the presence of DTG confer modest (3-5-fold) resistance to INSTIs, a level of resistance comparable to that observed with clinically relevant IN mutations. These NC mutations do not appreciably affect viral RNA packaging, viral assembly, virus release, or infectivity. qPCR and TOA analyses with RT inhibitors and INSTIs revealed that the NC mutations accelerate the kinetics of integration. Notably, the shortened time frame between reverse transcription and integration correlates with reduced sensitivity to DTG, suggesting that NC mutations limit the window of opportunity for INSTIs to bind intasomes and block integration. Examination of the sequence at 2-LTR junctions showed no significant difference, suggesting that NC mutants preserve viral DNA ends, which are important for integration and INSTI binding. During DTG selection in primary PBMCs, we identified NC-N27I and IN-S153Y, which conferred 2.5-fold and 3.4-fold resistance to DTG, respectively. We observed that mutations in NC and IN acted in concert to confer 8.5-fold resistance to DTG, suggesting that NC mutations engage in epistatic interactions with IN resistance mutations. Significantly, we identified some of the in vitro-selected NC mutations in PLWH who experienced VF with DTG-containing regimens.
Conclusions
These findings demonstrate that mutations in multiple regions in the HIV-1 genome – Env, NC, and IN – collectively contribute to INSTI resistance. These results further our understanding of INSTI resistance and support the need for genotypic analysis outside of IN in individuals failing INSTI-containing regimens.
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