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1.  The dual role of tetraspanin CD63 in HIV-1 replication 
Virology Journal  2014;11:23.
Previously, we showed that the tetraspanin membrane protein CD63 mediates both early and post-integration stages of the HIV-1 replication cycle. The temporal roles of CD63 were discerned using monoclonal antibodies and small interfering RNAs (siRNAs) to block CD63 function, and determining which of the sequential steps in HIV-1 replication were disrupted. Inhibition was shown to occur during early infection, suggestive of involvement in virus entry or reverse transcription. In addition, we have shown that treatment with CD63 siRNA post-infection, significantly inhibited virus production in supernatant, suggesting an important role for CD63 in macrophages during HIV-1 replication events occurring after proviral integration, and possibly during egress.
In this study we used CD63 siRNA to investigate the infectivity of pseudotyped viruses (carrying an NL4-3 Env-negative luciferase backbone) in primary human macrophages. We demonstrated that lab adapted R5- and R5X4-tropic HIV-1 strains are significantly inhibited by CD63 silencing. However, the infectivity of MLV or VSV-pseudotyped strains, which enter though receptor-mediated endocytosis, is unaffected by silencing CD63. These results indicate that CD63 may support Env-mediated entry or fusion events facilitated though CD4 and CCR5. Also, antibody and siRNA-based CD63 inhibition studies indicate a potential role for CD63 following proviral integration. Further, we show that CD63 expression is key for efficient replication in primary CD4+ T cells, complementing our prior studies with primary human macrophages and immortalized cell lines.
Collectively, these findings indicate that CD63 may support Env-mediated fusion as well as a late (post-integration) step in the HIV-1 replication cycle.
PMCID: PMC3944621  PMID: 24507450
HIV-1 replication cycle; Tetraspanin CD63; CD4+ T cells; Macrophages
2.  A Post-Entry Role for CD63 in Early HIV-1 Replication 
Virology  2011;412(2):315-324.
Macrophages and CD4+ lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4+ cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.
PMCID: PMC3060945  PMID: 21315401
HIV-1; Tetraspanin; CD63; siRNA; Macrophage; life cycle
3.  Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1 
AIDS Research and Human Retroviruses  2012;28(10):1329-1339.
Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.
PMCID: PMC3448097  PMID: 22404213
4.  A Functional Role for ADAM10 in Human Immunodeficiency Virus Type-1 Replication 
Retrovirology  2011;8:32.
Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1.
Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4+ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection.
We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.
PMCID: PMC3118345  PMID: 21569301
5.  Quantitative PCR used to Assess HIV-1 Integration and 2-LTR Circle Formation in Human Macrophages, Peripheral Blood Lymphocytes and a CD4+ Cell Line 
Virology Journal  2010;7:354.
Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL).
In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4+ cells by infecting with HIV-1SX (R5) or dual-tropic isolate HIV-189.6 (R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA.
These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.
PMCID: PMC3003270  PMID: 21129188
6.  Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus 
West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.
PMCID: PMC2809995  PMID: 20126583
West Nile virus (WNV); encephalitis; meningitis; double immunohistochemical staining; neurons; neuroglia
7.  Dried-Plasma Transport Using a Novel Matrix and Collection System for Human Immunodeficiency Virus and Hepatitis C Virus Virologic Testing▿  
Journal of Clinical Microbiology  2009;47(5):1491-1496.
A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log10 copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log10 copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.
PMCID: PMC2681841  PMID: 19321732
8.  Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility 
AIDS Research and Human Retroviruses  2008;24(10):1291-1300.
The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.
PMCID: PMC2721781  PMID: 18844463
9.  A Critical Role for CD63 in HIV Replication and Infection of Macrophages and Cell Lines 
Virology  2008;379(2):191-196.
HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.
PMCID: PMC2697030  PMID: 18682304
Tetraspanin; CD63; HIV-1; siRNA; macrophages
10.  Combinatorial Selection, Inhibition and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase† 
Biochemistry  2005;44(30):10388-10395.
Despite the key role played by the RNase H of Human Immunodeficiency Virus-1 Reverse Transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5′ end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from E.coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (Kd) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 μg/ml) markedly reduced viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 μg/ml to 2.0 μg/ml (IC50 <100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from multiplicity of infection (m.o.i) of 0.0005 to 0.05, with reduction of virus production by more than 50% at high m.o.i. Suppression of virus was comparable to that seen with AZT at m.o.i. ≦ 0.005.
PMCID: PMC2532674  PMID: 16042416
11.  Evolution of HIV Resistance Mutations in Patients Maintained on a Stable Treatment Regimen After Virologic Failure 
We compared the rate of emergence of thymidine analogue mutations (TAMs) and major protease inhibitor mutations in adherent patients who remained on stable treatment with a thymidine analogue and/or protease inhibitor after the onset of virologic failure.
Follow-up genotypic resistance testing was done using archived plasma obtained from patients having 0 or 1 TAM and/or 0 or 1 major protease inhibitor resistance mutation at the onset of virologic failure.
The median duration of observed failure was 691 days. There were 41 thymidine analogue regimens and 34 protease inhibitor regimens; concomitant ritonavir was used 4 times. New major protease inhibitor mutations emerged more rapidly than did new TAMs (P = 0.0019); new TAMs emerged more rapidly in thymidine analogue regimens that did not include lamivudine (P = 0.0073). The emergence of TAMs and major protease inhibitor mutations did not differ if lamivudine was not part of the thymidine analogue regimen. The evolution of CD4+ cell counts and plasma viral loads (pVLs) during virologic failure was similar regardless of whether or not a new TAM or major protease inhibitor mutations emerged or, for thymidine analogue–containing regimens, whether lamivudine was or was not used.
Major protease inhibitor mutations arose more frequently and rapidly than did TAMs in patients with sustained virologic failure who received lamivudine.
PMCID: PMC1832074  PMID: 17075391
HIV resistance; protease inhibitors; resistance mutations; reverse transcriptase inhibitors
12.  Potential Role for CD63 in CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection of Macrophages 
Journal of Virology  2003;77(6):3624-3633.
Macrophages and CD4+ lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or β-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.
PMCID: PMC149503  PMID: 12610138
13.  Cultivated Vaginal Microbiomes Alter HIV-1 Infection and Antiretroviral Efficacy in Colonized Epithelial Multilayer Cultures 
PLoS ONE  2014;9(3):e93419.
There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.
PMCID: PMC3968159  PMID: 24676219

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