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1.  Transcription of Preintegrated HIV-1 cDNA Modulates Cell Surface Expression of Major Histocompatibility Complex Class I via Nef ▿  
Journal of Virology  2011;85(6):2828-2836.
Although transcription from unintegrated human immunodeficiency virus type 1 (HIV-1) DNA can occur inside infected cells, yielding all classes of viral mRNA transcripts, the translation of viral proteins is very limited. One of the proteins made is Nef, but it is unclear whether Nef produced in this way is able to play a role in immune evasion as occurs with integrated virus. We therefore asked whether transcription from preintegrated HIV-1 cDNAs could result in Nef-mediated modulation of cell surface major histocompatibility complex class I (MHC-I) expression. We infected a Rev-CEM green fluorescent protein (GFP) reporter cell line with virus and blocked integration though use of either an inactive integrase or the integrase inhibitor raltegravir. Infected cells were assayed by flow cytometry for cell surface expression of the HLA-A, HLA-B, and HLA-C allotypes (HLA-ABC), HLA-A31, and HLA-E. Viral RNA and DNA products were assayed via quantitative PCR (qPCR). The prevention of integration had no effect, relative to productively infected cells, on levels of expression of multiply spliced viral mRNA transcripts and Nef protein. Downregulation of HLA-ABC and HLA-A31 also occurred at levels similar to those seen in cells in which integration had occurred. Parallel experiments assaying cell surface HLA-ABC expression in infected activated primary CD4+ T cells produced a similar pattern of results. Hence, the capacity of HIV-1 to modulate MHC-I is not linked to its ability to integrate. Thus, Nef-mediated evasion of host immune responsiveness might be attributable, in part at least, to transcription from unintegrated viral DNA.
doi:10.1128/JVI.01854-10
PMCID: PMC3067938  PMID: 21209113
2.  Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor ▿  
Journal of Virology  2010;84(18):9210-9216.
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 Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.
doi:10.1128/JVI.01164-10
PMCID: PMC2937597  PMID: 20610719
3.  Tetherin restricts direct cell-to-cell infection of HIV-1 
Retrovirology  2010;7:115.
Background
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.
Results
We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the influence of tetherin on cell-to-cell spread of HIV-1. Sup-T1 cells inducible for tetherin expression were used to examine the impact of effector and target cell tetherin expression on virus transfer and transmission. Using this assay, we showed that tetherin inhibits direct cell-to-cell virus transfer and transmission. Viral Vpu promoted viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface. Further, we showed that tetherin on the target cell promotes viral transfer and transmission. Viral infectivity in itself was not affected by tetherin.
Conclusion
In addition to inhibiting viral release, tetherin also inhibits direct cell-to-cell spread. Viral protein Vpu counteracts this restriction, outweighing its possible cost of fitness in cell-to-cell transmission. The differential role of tetherin in effector and target cells suggest a role for tetherin in cell-cell contacts and virological synapses.
doi:10.1186/1742-4690-7-115
PMCID: PMC3017029  PMID: 21184674
4.  The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity▿  
The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
doi:10.1128/AAC.01795-09
PMCID: PMC2876396  PMID: 20308384
5.  Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture▿  
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 provide in vitro evidence 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.
doi:10.1128/AAC.01537-09
PMCID: PMC2826010  PMID: 20038621
6.  Expression of Nef from unintegrated HIV-1 DNA downregulates cell surface CXCR4 and CCR5 on T-lymphocytes 
Retrovirology  2010;7:44.
Background
Transcription of HIV-1 cDNA prior to, or in the absence of, integration leads to synthesis of all classes of viral RNA transcripts. Yet only a limited range of viral proteins, including Nef, are translated in this context. Nef expression from unintegrated HIV-1 DNA has been shown to reduce cell surface CD4 levels in T-cells. We wished to determine whether Nef expressed from unintegrated DNA was also able to downregulate the chemokine coreceptors CXCR4 and CCR5.
Viral integration was blocked through use of an inactive integrase or by using the integrase inhibitor raltegravir. Infected cells bearing unintegrated DNA were assayed by flow cytometry in the GFP reporter cell line, Rev-CEM, for cell surface levels of CD4, CXCR4 and CCR5.
Results
In cells bearing only unintegrated HIV-1 DNA, we found that surface levels of CXCR4 were significantly reduced, while levels of CCR5 were also diminished, but not to the extent of CXCR4. We also confirmed the downregulation of CD4. Similar patterns of results were obtained with both integrase-deficient virus or with wild-type infections of cells treated with raltegravir. The Alu-HIV qPCR assay that we used for detection of proviral DNA did not detect any integrated viral DNA.
Conclusions
Our results demonstrate that Nef can be expressed from unintegrated DNA at functionally relevant levels and suggest a role for Nef in downregulation of CXCR4 and CCR5. These findings may help to explain how downregulation of CXCR4, CCR5 and CD4 might restrict superinfection and/or prevent signal transduction involving HIV-1 infected cells.
doi:10.1186/1742-4690-7-44
PMCID: PMC2881062  PMID: 20465832
7.  Human Immunodeficiency Virus Type 1 Recombinant Reverse Transcriptase Enzymes Containing the G190A and Y181C Resistance Mutations Remain Sensitive to Etravirine▿  
Antimicrobial Agents and Chemotherapy  2009;53(11):4667-4672.
Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.
doi:10.1128/AAC.00800-09
PMCID: PMC2772356  PMID: 19704127
8.  Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes 
Retrovirology  2009;6:103.
Background
Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes.
Methods
This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro. The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048.
Results
Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes.
Conclusion
This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.
doi:10.1186/1742-4690-6-103
PMCID: PMC2779801  PMID: 19906306
10.  An ATPase Activity Associated with the Rotavirus Phosphoprotein NSP5 
Virology  2007;369(2):389-399.
Interactions between NSP5 and NSP2 drive the formation of viroplasms, sites of genome replication and packaging in rotavirus-infected cells. The serine-threonine rich NSP5 transitions between hypo- and hyper-phosphorylated isomers during the replication cycle. In this study, we determined that purified recombinant NSP5 has a Mg2+-dependent ATP-specific triphosphatase activity that generates free ADP and Pi (Vmax of 19.33 fmol of product/min/pmol of enzyme). The ATPase activity was correlated with low levels of NSP5 phosphorylation, suggestive of a possible link between ATP hydrolysis and an NSP5 autokinase activity. Mutagenesis showed that the critical residue (Ser67) needed for NSP5 hyperphosphorylation by cellular casein kinase-like enzymes has no role in the ATPase or autokinase activities of NSP5. Through its NDP kinase activity, the NSP2 octamer may support NSP5 phosphorylation by creating a constant source of ATP molecules for the autokinase activity of NSP5 and for cellular kinases associated with NSP5.
doi:10.1016/j.virol.2007.07.029
PMCID: PMC2702534  PMID: 17825341
rotavirus; phosphoprotein; NSP5; ATPase
11.  Group A Human Rotavirus Genomics: Evidence that Gene Constellations Are Influenced by Viral Protein Interactions▿ †  
Journal of Virology  2008;82(22):11106-11116.
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.
doi:10.1128/JVI.01402-08
PMCID: PMC2573243  PMID: 18786998

Results 1-11 (11)