Background:
We reported that mutations in the envelope glycoprotein (Env) can broadly reduce HIV-1 susceptibility to ARVs. The aim of the current study was to examine the pathway(s) by which HIV-1 develops high-level resistance to the integrase (IN) strand transfer inhibitor (INSTI) dolutegravir (DTG).
Methods:
Long-term passaging of lab-adapted and primary viral isolates using the SupT1 T-cell line was performed over nearly one year with an escalating concentration (0.1 – 2,000 nM) of DTG. Viral sequence analysis was performed longitudinally. Identified mutations were introduced into WT HIV-1 molecular clones and the replication kinetics and viral infection through cell-cell contact were examined in the presence and absence of drug. To measure the multiplicity of infection (MOI), we monitored viral replication by co-infection with eGFP- and mRuby-expressing reporter viruses harboring the Env mutations.
Results:
In a manner independent of viral isolate and coreceptor usage, HIV-1 became resistant to DTG by sequentially acquiring mutations in Env and Gag-nucleocapsid (NC) in the absence of resistance mutations in IN. The selected NC mutations clustered in the zinc-finger domain and conferred modest (3-5 fold) resistance to INSTIs. An Env mutant, 7XEnv, containing seven substitutions (V85A, S162K, R298K, Q363R, A541V, V693I and G825E) exhibited faster-than-WT replication and resistance to multiple classes of ARVs, with the fold resistance being markedly higher for INSTIs. Viral transmission of 7XEnv through cell-cell contact is 15-fold more efficient than WT, resulting in a higher MOI and reduced sensitivity to DTG. Viral infection using VSV-G-pseudotyped viruses over a range of MOIs revealed that INSTIs are more readily overwhelmed by high MOI compared to RT inhibitors. Co-infection experiments using fluorescently tagged reporter viruses demonstrated that 7XEnv infection leads to a higher number of cells expressing multiple proviruses compared to WT.
Conclusions:
These findings demonstrate that a combination of mutations in Env and NC can confer high-level resistance to INSTIs in the absence of IN mutations. The Env mutations overcome inhibition by INSTIs through increased MOI mediated by highly efficient cell-cell transfer. These results advance the understanding of how HIV-1 can evolve resistance to ARVs in the absence of mutations in drug-target genes and provide new insights into the contribution of cell-cell transfer to viral replication and drug resistance.