Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication.

Significant intra- and interindividual variability has been observed in response to use of pharmacological agents in treatment of HIV infection. Treatment of HIV infection is limited by high rates of adverse drug reactions and development of resistance in a significant proportion of patients as a result of suboptimal drug concentrations. The efficacy of antiretroviral therapy is challenged by the emergence of resistant HIV-1 mutants with reduced susceptibility to antiretroviral drugs. Moreover, pharmacotherapy of patients infected with HIV is challenging because a great number of comorbidities increase polypharmacy and the risk for drug-drug interactions. Drug-metabolizing enzymes and drug transporters regulate drug access to the systemic circulation, target cells, and sanctuary sites. These factors, which determine drug exposure, along with the emergence of mutations conferring resistance to HIV medications, could explain variability in efficacy and adverse drug reactions associated with antiretroviral drugs. In this review, the major factors affecting the disposition of antiretroviral drugs, including key drug-metabolizing enzymes and membrane drug transporters, are outlined. Genetic polymorphisms affecting the activity and/or the expression of cytochromes P450 or UGT isozymes and membrane drug transport proteins are highlighted and include such examples as the association of neurotoxicity with efavirenz, nephrotoxicity with tenofovir, hepatotoxicity with nevirapine, and hyperbilirubinemia with indinavir and atazanavir. Mechanisms of drug resistance conferred by specific viral mutations are also reviewed, with particular attention to replicative viral fitness and transmitted HIV drug resistance with the objectives of providing a better understanding of mechanisms involved in HIV drug resistance and helping health care providers to better manage interpatient variability in drug efficacy and toxicity.

Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN)-inducible factor of the innate immune system, recently shown to exert antiviral activity against HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface, thereby inhibiting viral release. In HIV-1 infection, the viral protein U (Vpu) counteracts this antiviral action by down-modulating tetherin from the cell surface. Viral dissemination between T-cells can occur via cell-free transmission or the more efficient direct cell-to-cell route through lipid raft-rich virological synapses, to which tetherin localizes.

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg ²⁺ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

Background: Because of high intersubtype HIV-1 genetic variability, it has been shown that subtype-specific patterns of resistance to antiretroviral drugs exist. We wished to ascertain whether this might be true for integrase inhibitors. Methods: We compared the susceptibility of subtype B and C HIV-1 integrase enzymes, harboring the previously reported resistance mutations E92Q, N155H, and E92Q/N155H, to clinically relevant integrase inhibitors. This was performed biochemically using a microtiter plate system. Results: Subtype C integrase enzymes bearing the resistance mutations E92Q/N155H were approximately 10-fold more susceptible to each of two integrase inhibitors, raltegravir and elvitegravir, than were subtype B recombinant integrase containing the same mutations. Conclusion: Polymorphic differences within the subtype B and C integrase genes likely cause variations in the contribution of N155H alone or in combination with E92Q to drug resistance. It is possible that different viral subtypes may favor different mutational pathways, potentially leading to varying levels of drug resistance among different subtypes.

Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data providein vitroevidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.