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1.  Detailed Topology Mapping Reveals Substantial Exposure of the “Cytoplasmic” C-Terminal Tail (CTT) Sequences in HIV-1 Env Proteins at the Cell Surface 
PLoS ONE  2013;8(5):e65220.
Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20–50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.
PMCID: PMC3664582  PMID: 23724133
2.  Comparison of the Effects of Pathogenic Simian Human Immunodeficiency Virus Strains SHIV-89.6P and SHIV-KU2 in Cynomolgus Macaques 
Factors explaining why human immunodeficiency virus (HIV) enhances the risk of reactivated tuberculosis (TB) are poorly understood. Unfortunately, experimental models of HIV-induced reactivated TB are lacking. We examined whether cynomolgus macaques, which accurately model latent TB in humans, could be used to model pathogenesis of HIV infection in the lungs and associated lymph nodes. These experiments precede studies modeling the effects of HIV infection on latent TB. We infected two groups of macaques with chimeric simian–human immunodeficiency viruses (SHIV-89.6P and SHIV-KU2) and followed viral titers and immunologic parameters including lymphocytes numbers and phenotype in the blood, bronchoalveolar lavage cells, and lymph nodes over the course of infection. Tissues from the lungs, liver, kidney, spleen, and lymph nodes were similarly examined at necropsy. Both strains produced dramatic CD4+ T cell depletion. Plasma titers were not different between viruses, but we found more SHIV-89.6P in the lungs. Both viruses induced similar patterns of cell activation markers. SHIV-89.6P induced more IFN-γ expression than SHIV-KU2. These results indicate SHIV-89.6P and SHIV-KU2 infect cynomolgus macaques and may be used to accurately model effects of HIV infection on latent TB.
PMCID: PMC3311977  PMID: 18366326
3.  Topology of the C-Terminal Tail of HIV-1 gp41: Differential Exposure of the Kennedy Epitope on Cell and Viral Membranes 
PLoS ONE  2010;5(12):e15261.
The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the “intracytoplasmic domain” based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical “Kennedy epitope” (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.
PMCID: PMC2998427  PMID: 21151874
4.  Reactivation of Latent Tuberculosis in Cynomolgus Macaques Infected with SIV Is Associated with Early Peripheral T Cell Depletion and Not Virus Load 
PLoS ONE  2010;5(3):e9611.
HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.
PMCID: PMC2835744  PMID: 20224771
5.  Differential Functional Phenotypes of Two Primary HIV-1 Strains Resulting from Homologous Point Mutations in the LLP Domains of the Envelope gp41 Intracytoplasmic Domain 
Virology  2007;367(1):102-116.
We previously reported that selected mutations of highly conserved arginine residues within the LLP regions of HIV-1ME46 gp41 had diverse effects on Env function. In the current study, we sought to test if the observed LLP mutant phenotypes would be similar in HIV-189.6. The results of the current studies revealed that the LLP-1 mutations conferred reduced Env incorporation, infectivity, and replication phenotypes in both viruses, while homologous LLP-2 mutations had differential phenotypical effects between the two strains. In particular, several of the 89.6 LLP-2 mutant viruses were replication defective in CEMX174 cells despite having increased levels of Env incorporation, and with both strains, there were differential effects on infectivity. This comparison of homologous point mutations in two different strains of HIV supports the role of LLPs as determinants of Env function, but reveals for the first time the influence of virus strain on LLP mutant phenotypes.
PMCID: PMC2034414  PMID: 17582453
Site-directed mutagenesis; Cell fusion; HIV envelope protein gp41; HIV; Viral infectivity; Virus Replication; HIV envelope incorporation and biosynthesis
Vaccine  2006;25(5):834-845.
We previously reported that an experimental live-attenuated equine infectious anemia virus (EIAV) vaccine, containing a mutated S2 accessory gene, provided protection from disease and detectable infection after virulent virus (EIAVPV) challenge [1,2]. To determine if attenuated EIAV vaccines actually prevent persistent infection by challenge virus, we employed a 14-day dexamethasone treatment of vaccinated horses post-challenge to suppress host immunity and amplify replication levels of any infecting EIAV. At two months post-challenge the horses were all protected from virulent-virus challenge, evidenced by a lack of EIA signs and detectable challenge plasma viral RNA. Upon immune suppression, 6/12 horses displayed clinical EIA. Post-immune suppression characterizations demonstrated that the attenuated vaccine evidently prevented detectable challenge virus infection in 50% of horses. These data highlight the utility of post-challenge immune suppression for evaluating persistent viral vaccine protective efficacy.
PMCID: PMC1855206  PMID: 17023099
EIAV; Vaccine; Live-Attenuated; Immune Suppression; Dexamethasone

Results 1-6 (6)