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1.
J Virol ; 89(1): 833-43, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25355888

ABSTRACT

UNLABELLED: Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity. IMPORTANCE: Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.


Subject(s)
Drug Resistance, Viral , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-2/enzymology , HIV-2/physiology , Mutation, Missense , Virus Replication , Amino Acid Substitution , HIV Reverse Transcriptase/chemistry , HIV-2/genetics , Humans , Molecular Dynamics Simulation
2.
PLoS One ; 8(10): e74078, 2013.
Article in English | MEDLINE | ID: mdl-24098331

ABSTRACT

Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume.


Subject(s)
HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/drug effects , HIV-1/enzymology , Polymorphism, Genetic , Reverse Transcriptase Inhibitors/pharmacology , Ribonuclease H, Human Immunodeficiency Virus/metabolism , Drug Resistance, Viral/genetics , Genomics , HIV Reverse Transcriptase/genetics , HIV-1/physiology , Molecular Dynamics Simulation , Protein Structure, Tertiary , Virus Replication/drug effects , Virus Replication/genetics
3.
J Clin Microbiol ; 49(4): 1280-6, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21307214

ABSTRACT

The incidence of simian virus 40 (SV40) infections in rhesus macaques infected with simian-human immunodeficiency viruses (SHIV) and in uninfected animals was determined using PCR. Rates varied from 5% in peripheral blood mononuclear cells of uninfected monkeys to 19.6% in SHIV-infected macaques. Much higher detection rates, up to 75%, were found in lymph nodes and spleen samples of SHIV-infected animals. Sequence analysis of PCR amplicons revealed that they form two genetic clusters, one containing the majority of known SV40 strains and the other formed by variants with 7% genetic difference. Based on this difference, we propose two SV40 types: "type 1" or "classical type" for the majority of SV40 strains and "type 2" for the novel SV40 variants. The genome of one variant, SV40-Ri257, was completely sequenced and analyzed. The agnogene of SV40-Ri257 extends into the VP2 open reading frame and encodes a typical agnoprotein fused to a C-terminal hydrophobic region. The transcriptional control region (TCR) of SV40-Ri257 is the least conserved region compared to type 1 viruses. Particularly, the 3' end of the TCR, containing the early promoter and enhancer region, exhibits considerable variation. Further analysis of SHIV-infected macaques with type-specific PCRs revealed that the TCR of type 1 was completely conserved, whereas this region in type 2 varied considerably within the early enhancer region. We provide evidence here for the existence of a novel SV40 type in rhesus macaques and show that double infections with both types frequently occur.


Subject(s)
Polyomavirus Infections/veterinary , Primate Diseases/epidemiology , Primate Diseases/virology , Simian virus 40/classification , Simian virus 40/isolation & purification , Tumor Virus Infections/veterinary , Animals , Blood/virology , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , Incidence , Leukocytes, Mononuclear/virology , Lymph Nodes/virology , Macaca mulatta , Molecular Sequence Data , Phylogeny , Polyomavirus Infections/epidemiology , Polyomavirus Infections/virology , Sequence Analysis, DNA , Simian virus 40/genetics , Spleen/virology , Tumor Virus Infections/epidemiology , Tumor Virus Infections/virology
4.
Virol J ; 7: 347, 2010 Nov 26.
Article in English | MEDLINE | ID: mdl-21110837

ABSTRACT

The complete nucleotide sequences of three chimpanzee polyomavirus genetic variants were determined. Phylogenetic analysis indicated that the viruses form two different genotypes of ChPyV. Comparison with other primate polyomaviruses revealed a putative agnogene, and an unusually long VP1 open reading frame. The transcriptional control regions (TCR) of the viruses were extremely short (155 nucleotides), and highly conserved amongst the genotypes. Analysis of the TCR from different chimpanzee subspecies, and from a series of tissues from five individuals confirmed its genetic stability, and also indicates that double-infections with different genotypes can occur.


Subject(s)
Pan troglodytes/virology , Polyomavirus/classification , Polyomavirus/isolation & purification , Animals , Cluster Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Female , Genotype , Male , Molecular Sequence Data , Phylogeny , Polyomavirus/genetics , Sequence Analysis, DNA , Transcription, Genetic , Viral Proteins/genetics
5.
Glia ; 55(15): 1589-600, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17823968

ABSTRACT

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.


Subject(s)
Antigen-Presenting Cells/physiology , Microglia/physiology , Toll-Like Receptor 8/physiology , Animals , Antigen-Presenting Cells/immunology , Bone Marrow Cells/drug effects , Cell Differentiation/physiology , Cell Lineage/physiology , Cell Proliferation , Cell Separation , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Lymphocyte Culture Test, Mixed , Macaca mulatta , Macrophage Activation/physiology , Macrophage Colony-Stimulating Factor/pharmacology , Male , Microglia/immunology , Phagocytosis/drug effects , Phagocytosis/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 8/biosynthesis , Toll-Like Receptor 8/genetics
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