Genotypic characterization of gag-pol cleavage site mutations in HIV-1 infected patients failing HAART
Date
2014-04-02
Authors
Ramatsebe, Majoalane Tina Maria
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Abstract
Sequence analysis from HIV-1 (human immunodeficiency virus type 1) subtype B and more
recently subtype C infected patients has revealed that mutations in the HIV-1 protease region
that confer drug resistance to boosted protease inhibitor (PIs) are rarely detected at the time
of virological failure. Mutations in the HIV-1 subtype B gag-pol cleavage sites are thought to
be compensatory mutations which arise as a result of PI use. This study investigated the
presence of compensatory mutations in the HIV-1 subtype C gag-pol cleavage sites and
matched pol genotypes from South African patients failing a boosted PI-based regimen, as
compared to antiretroviral drug naïve patients.
A new amplification protocol encompassing the near full-length gag, PR and partial RT was
established and used to sequence the HIV-1 gag-pol cleavage sites from 23 proviral DNA
samples (p24 antigen cultured peripheral blood mononuclear cells; PBMCs), and 51 patient
samples (23 antiretroviral drug-naïve, 26 failing second-line lopinavir/ritonavir containing
regimens), all attending the Charlotte Maxeke Johannesburg Hospital. Nucleotide sequences
were aligned and codon positions S373Q, A431V, I437T/V, L449P or P453L associated with
known gag-pol cleavage site mutations were analysed and compared. The pol genotypes were
established using an in house assay. Antiretroviral drug resistant primary virus isolates were
grown from samples from patients enrolled on the CIPRA-SA study, and propagated in coculture
with PHA-activated, IL-2 stimulated PBMCs. HIV-1 gag-pol cleavage sites and pol
genotypes for all primary virus isolates were established as described above.
Fifty one of 74 patient samples, used to establish the in-house gag-pol cleavage site assay,
were successfully amplified and sequenced. Detailed analysis of the five known gag-pol
cleavage sites revealed that 5 patient samples (4 PI-exposed, 1 unknown regimen) encoded
for the previously described mutations that impact on gag-pol cleavage in the absence of any
major PR mutations. A further five samples from patients on the failing PI-based regimen had
major PR mutations. No known mutations in the gag-pol region were identified in patients
failing a first line regimen. The pol mutations described in this study were similar to the
findings reported for treatment failures in South African HIV-1 subtype C infected patients.
Primary virus was grown from only 25 of the 91 PBMC CIPRA samples. None of the 25
CIPRA-SA primary virus isolates had gag-pol cleavage site mutations, and only 9 harboured
known RT antiretroviral drug resistant mutations.
Overall, the presence of HIV-1 gag-pol cleavage site mutations may account for virological
treatment failure in 5 of the South African patient samples analysed. Although the gag-pol
cleavage site mutations detected in the current study are only present in a small proportion of
treatment-experienced South African patients, this may increase due to more patients
accessing second line PI-containing regimens. Thus, future genotyping work incorporating
the analysis of the gag-pol cleavage sites in addition to the PR and RT regions is warranted.
The antiretroviral drug resistant primary viruses obtained provide valuable reagents for future
phenotyping studies.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand , in fulfillment of the requirements for the degree of Master of Science in Medicine, 2013