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1.  Dynamics of HIV Type 1 Recombination Following Superinfection 
Abstract
There are currently few detailed studies describing HIV-1 recombination events or the potential impact of recombination on drug resistance. We describe here the viral recombination dynamics in a drug-naive patient initially infected with a circulating recombinant form 19 (CRF19) virus containing transmitted drug resistance mutations followed by superinfection with “wild-type” subtype B virus. Single genome analysis showed replacement of the primary CRF19 virus by recombinants of the CRF19 virus and the superinfecting subtype B virus. The CRF19/B recombinant virus dominating after superinfection had lost drug resistance mutations and at no time was the superinfecting subtype B variant found to be dominant in blood plasma. Furthermore, the detection of recombinant viruses in seminal plasma indicates the potential for onward transmission of these strains.
doi:10.1089/aid.2013.0009
PMCID: PMC3653373  PMID: 23495713
2.  Phylodynamic and Phylogeographic Patterns of the HIV Type 1 Subtype F1 Parenteral Epidemic in Romania 
Abstract
In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981–1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.
doi:10.1089/aid.2011.0390
PMCID: PMC3423652  PMID: 22251065
3.  Low frequency of genotypic resistance in HIV-1-infected patients failing an atazanavir-containing regimen: a clinical cohort study 
Dolling, David I. | Dunn, David T. | Sutherland, Katherine A. | Pillay, Deenan | Mbisa, Jean L. | Parry, Chris M. | Post, Frank A. | Sabin, Caroline A. | Cane, Patricia A. | Aitken, Celia | Asboe, David | Webster, Daniel | Cane, Patricia | Castro, Hannah | Dunn, David | Dolling, David | Chadwick, David | Churchill, Duncan | Clark, Duncan | Collins, Simon | Delpech, Valerie | Geretti, Anna Maria | Goldberg, David | Hale, Antony | Hué, Stéphane | Kaye, Steve | Kellam, Paul | Lazarus, Linda | Leigh-Brown, Andrew | Mackie, Nicola | Orkin, Chloe | Rice, Philip | Pillay, Deenan | Phillips, Andrew | Sabin, Caroline | Smit, Erasmus | Templeton, Kate | Tilston, Peter | Tong, William | Williams, Ian | Zhang, Hongyi | Zuckerman, Mark | Greatorex, Jane | Wildfire, Adrian | O'Shea, Siobhan | Mullen, Jane | Mbisa, Tamyo | Cox, Alison | Tandy, Richard | Hale, Tony | Fawcett, Tracy | Hopkins, Mark | Ashton, Lynn | Booth, Claire | Garcia-Diaz, Ana | Shepherd, Jill | Schmid, Matthias L. | Payne, Brendan | Hay, Phillip | Rice, Phillip | Paynter, Mary | Bibby, David | Kirk, Stuart | MacLean, Alasdair | Gunson, Rory | Coughlin, Kate | Fearnhill, Esther | Fradette, Lorraine | Porter, Kholoud | Ainsworth, Jonathan | Anderson, Jane | Babiker, Abdel | Fisher, Martin | Gazzard, Brian | Gilson, Richard | Gompels, Mark | Hill, Teresa | Johnson, Margaret | Kegg, Stephen | Leen, Clifford | Nelson, Mark | Palfreeman, Adrian | Post, Frank | Sachikonye, Memory | Schwenk, Achim | Walsh, John | Huntington, Susie | Jose, Sophie | Thornton, Alicia | Glabay, Adam | Orkin, C. | Garrett, N. | Lynch, J. | Hand, J. | de Souza, C. | Fisher, M. | Perry, N. | Tilbury, S. | Gazzard, B. | Nelson, M. | Waxman, M. | Asboe, D. | Mandalia, S. | Delpech, V. | Anderson, J. | Munshi, S. | Korat, H. | Welch, J. | Poulton, M. | MacDonald, C. | Gleisner, Z. | Campbell, L. | Gilson, R. | Brima, N. | Williams, I. | Schwenk, A. | Ainsworth, J. | Wood, C. | Miller, S. | Johnson, M. | Youle, M. | Lampe, F. | Smith, C. | Grabowska, H. | Chaloner, C. | Puradiredja, D. | Walsh, J. | Weber, J. | Ramzan, F. | Mackie, N. | Winston, A. | Leen, C. | Wilson, A. | Allan, S. | Palfreeman, A. | Moore, A. | Wakeman, K.
Journal of Antimicrobial Chemotherapy  2013;68(10):2339-2343.
Objectives
To determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir.
Methods
Resistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list.
Results
Four hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84–3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience.
Conclusions
Viral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented.
doi:10.1093/jac/dkt199
PMCID: PMC3772741  PMID: 23711895
HIV; drug resistance mutations; naive patients; protease inhibitors; virological failure
4.  Patterns of resistance development with integrase inhibitors in HIV 
Raltegravir, the only integrase (IN) inhibitor approved for use in HIV therapy, has recently been licensed. Raltegravir inhibits HIV-1 replication by blocking the IN strand transfer reaction. More than 30 mutations have been associated with resistance to raltegravir and other IN strand transfer inhibitors (INSTIs). The majority of the mutations are located in the vicinity of the IN active site within the catalytic core domain which is also the binding pocket for INSTIs. High-level resistance to INSTIs primarily involves three independent mutations at residues Q148, N155, and Y143. The mutations significantly affect replication capacity of the virus and are often accompanied by other mutations that either improve replication fitness and/or increase resistance to the inhibitors. The pattern of development of INSTI resistance mutations has been extensively studied in vitro and in vivo. This has been augmented by cell-based phenotypic studies and investigation of the mechanisms of resistance using biochemical assays. The recent elucidation of the structure of the prototype foamy virus IN, which is closely related to HIV-1, in complex with INSTIs has greatly enhanced our understanding of the evolution and mechanisms of IN drug resistance.
doi:10.2147/IDR.S7775
PMCID: PMC3108751  PMID: 21694910
raltegravir; elvitegravir; integrase inhibitors; HIV; drug resistance
5.  The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains 
Retrovirology  2011;8:31.
Background
The Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.
Results
Single-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.
Conclusions
We demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.
doi:10.1186/1742-4690-8-31
PMCID: PMC3113953  PMID: 21569325
6.  Intrapatient Variation of the Respiratory Syncytial Virus Attachment Protein Gene▿  
Journal of Virology  2010;84(19):10425-10428.
Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.
doi:10.1128/JVI.01181-10
PMCID: PMC2937772  PMID: 20660195
7.  APOBEC3F and APOBEC3G Inhibit HIV-1 DNA Integration by Different Mechanisms▿  
Journal of Virology  2010;84(10):5250-5259.
APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3′ long terminal repeat (3′-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3′ processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3′-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.
doi:10.1128/JVI.02358-09
PMCID: PMC2863843  PMID: 20219927
8.  Human Immunodeficiency Virus Type 1 cDNAs Produced in the Presence of APOBEC3G Exhibit Defects in Plus-Strand DNA Transfer and Integration▿ †  
Journal of Virology  2007;81(13):7099-7110.
Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.
doi:10.1128/JVI.00272-07
PMCID: PMC1933301  PMID: 17428871
9.  Mutations in the RNase H Primer Grip Domain of Murine Leukemia Virus Reverse Transcriptase Decrease Efficiency and Accuracy of Plus-Strand DNA Transfer 
Journal of Virology  2005;79(1):419-427.
The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either β-galactosidase (lacZ) or green fluorescent protein (GFP) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.
doi:10.1128/JVI.79.1.419-427.2005
PMCID: PMC538714  PMID: 15596835

Results 1-9 (9)