Background Although drug resistance is a major challenge in HIV therapy the effect of drug resistance mutations about HIV evolution in vivo is not well comprehended. 109 cloned PR-RT sequences and that the majority of mutations were related to drug resistance. Moreover the PBMCs included archival varieties that reflected the treatment history of the patient while those in plasma were mainly related to the CP-868596 most recent treatment. Some of the proviral clones contained solitary or multiple mutations in various mixtures. Approximately eighteen percent of the proviral clones derived from infected PBMCs were defective i.e. 5.5% contained single nucleotide deletions (frameshift mutations) and 12.8% encoded in-frame quit codons (nonsense mutations). Amino acid substitutions in PR and the polymerase region of RT occurred in 12-15% of instances but were much less frequent in the RNase H region of RT which might not have been under drug selection pressure. Summary Selective drug pressure can yield multiple drug-resistant quasispecies that include archival and replication-incompetent varieties in PBMC reservoirs. Findings HIV quasispecies within infected individuals can rapidly adapt to hosts [1-7] due in part to variations in replicative fitness that enable some viruses to grow faster than others[3 8 This is of obvious medical relevance since viral genetic changes can result in alterations in receptor utilization escape from drug and host immune pressure and may impact on viral pathogenesis[9]. HIV-1 may also evolve separately in different physiological compartments e.g. peripheral blood mononuclear cells (PBMCs) vs. the central nervous system[10]. Here we statement on a person who failed multiple antiviral therapies (ART) including use of nucleoside and non-nucleoside RT inhibitors (NRTIs and NNRTIs) and protease inhibitors (PIs). After initiating therapy elsewhere with undisclosed regimens the patient was treated in 1999 in the Jewish General Hospital Montreal Canada with zidovudine (AZT)/lamivudine (3TC)/efavirenz (EFV) plus unboosted indinavir (IDV) and nelfinavir (NFV) for 9 weeks and 3 months respectively and was switched to stavudine (d4T)/3TC/amprenavir (APV) for 12 months at which time viral samples were obtained for resistance screening. Viral RNA from plasma and proviral DNA from PBMCs were purified using commercial packages (Qiagen Mississauga ON Canada). Initial HIV-1 genotyping was performed using Trugene HIV-1 genotyping packages (Siemens Diagnostics Inc. Toronto Canada). All analyzed were performed with authorization of the Ethics Review Committee Jewish General Hospital. The degree of quasispecies heterogeneity was higher in PBMCs than in plasma Mutations in PR and RT associated with drug resistance were compared in plasma vs PBMCs. Both types of samples contained viruses with multiple main CP-868596 (M46I/L G48V I54V V82A or L90M) and secondary resistance mutations (e.g. L10I) in PR as well as multiple mutations in RT (M41L E44A T69N V118I Mouse monoclonal to S1 Tag. S1 Tag is an epitope Tag composed of a nineresidue peptide, NANNPDWDF, derived from the hepatitis B virus preS1 region. Epitope Tags consisting of short sequences recognized by wellcharacterizated antibodies have been widely used in the study of protein expression in various systems. M184V L210W T215Y K219R for NRTIs) (A98G K101E V1081 Y181C and G190A for NNRTIs) (Table ?(Table1).1). Both the plasma and PBMC samples contained mixtures of mutations although some mutational motifs were only recognized in CP-868596 the PBMCs. For example mixtures of 41K/R 54 64 82 90 in PR and 181Y/C 190 219 in RT were recognized in PBMCs but not in plasma. Conversely 35 and 69N in PR and 108I in RT were detected only in plasma but not PBMCs as determined by genotyping. These results were confirmed by clonal sequencing of PBMC DNA. In general viruses harbouring the unboosted protease motif including L90M were exclusively present in PBMCs. This is consistent with the fact that genotyping often fails to detect minority varieties that are displayed at levels <10 to 35% in a given population [11]. Table 1 Comparisons of plasma and PBMC genotypes of CP-868596 the PR and RT areas in the HIV-1 infected patient Position in PR or RTa Resistance-associated mutations in PR-RT clones reveal heterogeneous viral populations within infected PBMCs Viral genetic diversity in the infected PBMCs was analyzed by randomly selecting and sequencing 109 clones of two self-employed cloning attempts. Nested PCR was performed to amplify the entire PR-RT region. One pair of primers ahead 5'-ACTGAGAGACAGGCTAATTTTTTAGG and backward 5'-TTGGGCCTTATCTATTTCCAT (Bio S&T Montreal.