Susceptibility Measurements Using Resistance Test Vectors with or without Patient-Derived C-terminus of RT, RNAseH and Integrase Are Largely Concordant

Currently available drug susceptibility assays are limited to protease (PR) and reverse transcriptase (RT), and do not capture C-terminal regions of RT, RNaseH or integrase (IN).  Here we describe a modification of a single cycle replication assay (PhenoSense HIV) that enables evaluation of IN inhibitors using a patient derived amplicon containing the entire polymerase (pol) gene.

Resistance test vectors (RTVs) containing the entire pol gene from 27 patient viruses were constructed.  Susceptibility results (fold change in IC₅₀ or FC) were compared to RTVs containing the standard PR/RT amplicon (codons 1-305 of RT) from the same samples. 

Susceptibility to PR inhibitors (PIs), nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) was measured using the PR/RT and pol RTVs.  Effects of IN, RNaseH, or C-terminal regions of RT were investigated by exchanging different domains of pol.

Results

Overall there was no difference in susceptibility to PIs, NRTIs or NNRTIs between the 2 types of RTV (t test, p>0.3).  In most cases there was no change in classification i.e. hyper-susceptible (FC < 0.4), sensitive, or reduced susceptibility (FC > 2.5). 

Only one sample showed reduced susceptibility (NVP FC 2.8) with the pol RTV not detected with PRRT (FC 0.3).  One patient virus with NNRTI resistance (K103N, P225H; wild-type at other NNRTI positions including 318) exhibited 6- to10-fold higher FC to NNRTIs when the pol-based RTV was used (e.g. EFV FC 202-fold vs. 20-fold). 

Exchange of different domains of pol indicated that the C-terminus of RT (after codon 305) and/or RNaseH are essential for this increased resistance.  Unique mutations in the C-terminus of RT of this sample are being investigated.

Conclusions

With rare exceptions, concordant measurements were obtained with recombinant viruses that express either the entire patient derived pol or only PR and incomplete RT fragments.  In one sample, where enhanced NNRTI resistance was observed, molecular analysis revealed that domains in pol after codon 305 were essential for this resistance.  Capturing the entire patient pol gene has the potential to retain protein-protein interactions from the native virus. Novel mutations within this region could induce enhanced NNRTI resistance by causing structural changes in the NNRTI-binding pocket of RT.

06/27/05

Reference
S Gupta and others. Susceptibility Measurements Using Resistance Test Vectors with or without Patient-Derived C-terminus of RT, RNAseH and Integrase are Largely Concordant (oral). Abstract 81. XIV International Drug Resistance Workshop. June 7-10, 2005. Quebec City, Quebec, Canada. [Antiviral Therapy 2005; 10:S91]

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