Tenofovir (TDF)-selected or abacavir (ABC)-selected low-frequency HIV type 1 subpopulations during failure with persistent viremia as detected by ultradeep pyrosequencing.

Detection of drug resistance is critical for determining antiretroviral treatment options. Ultradeep pyrosequencing (UDPS; 454 Life Sciences) is capable of detecting virus variant subpopulations with much greater sensitivity than population sequencing, which typically has a detection limit around 20%. UDPS of the HIV-1 reverse transcriptase (RT) (amino acids 56-120) was performed to detect the key mutations K65R and L74V associated with tenofovir and abacavir use. Plasma specimens from subjects with persistent rebound viremia following suppression on tenofovir (n = 8) or abacavir (n = 9)-based therapy were studied. Samples from a subject treated with zidovudine/lamivudine/efavirenz with a similar loss of virologic response served as a control. HIV-1 plasma RNA was ≥3.68 log(10) copies/ml at all time points sequenced. The median number of UDPS sequences analyzed/time point was 33,246. Among the eight tenofovir-treated subjects, three showed high-frequency (>20%) RT K65R at the time of failure, whereas one showed low-frequency (<20%) L74V; no low-frequency K65R was detected in these subjects. Among the nine abacavir-treated subjects, three showed low-frequency K65R; no L74V was detected in these patients. No K65R or L74V was detected in the samples from the control subject. At failure, other RT mutations were detected, including low-frequency NNRTI-resistant species detected at ≥1 time point in nine subjects; the key NNRTI mutation K103N, however, was always observed at >20% frequency. Although UDPS is useful in the detection of low-frequency subpopulations with transmitted resistance in antiviral-naive patients, it may have less utility in treatment-experienced patients with persistent viremia on therapy.

Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication.

Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IkappaBalpha required to activate NF-kappaB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IkappaBalpha depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IkappaBalpha depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-kappaB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.

Effect of tenofovir subtraction on HIV plasma viraemia, CD4+ T-cell count and resistance in a patient with baseline K65R and M184V mutations.

In vitro, the reverse transcriptase mutation K65R can simultaneously reduce drug susceptibility, replicative capacity and restrict HIV-1 replication. Here, we assessed the effect of tenofovir discontinuation for a patient receiving antiretroviral therapy whose HIV-1 had a dominant K65R/M184V genotype. Although limited by the single-case nature, the data support a hypothesis that there is no HIV viral RNA or CD4+ T-cell count benefit of taking tenofovir for experienced patients with genotypic evidence of K65R/M184V.