scholarly journals An In Vitro Rapid-Turnover Assay for Human Immunodeficiency Virus Type 1 Replication Selects for Cell-to-Cell Spread of Virus

2000 ◽  
Vol 74 (23) ◽  
pp. 10882-10891 ◽  
Author(s):  
Suryaram Gummuluru ◽  
C. Mathew Kinsey ◽  
Michael Emerman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mitomycin C ◽  
Culture System ◽  
Cell Transfer ◽  
Rapid Turnover ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Cell To Cell Transfer

ABSTRACT We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in the vpu open reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the Δvpu virus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the Δvpu virus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill?

2005 ◽  
Vol 79 (21) ◽  
pp. 13579-13586 ◽  
Author(s):  
W. David Wick ◽  
Otto O. Yang ◽  
Lawrence Corey ◽  
Steven G. Self
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.

scholarly journals Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

2010 ◽  
Vol 84 (23) ◽  
pp. 12185-12199 ◽  
Author(s):  
Clare Jolly ◽  
Nicola J. Booth ◽  
Stuart J. D. Neil
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Surface ◽  
Cell Transfer ◽  
Free Virion ◽  
Immunodeficiency Virus ◽  
Cell To Cell Transfer ◽  
Cell Spread ◽  
Hiv 1

ABSTRACT Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4+ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.

scholarly journals The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 77 (23) ◽  
pp. 12507-12522 ◽  
Author(s):  
Sébastien Violot ◽  
Saw See Hong ◽  
Dina Rakotobe ◽  
Caroline Petit ◽  
Bernard Gay ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dependent Manner ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

scholarly journals Cholesterol-Depleting Statin Drugs Protect Postmitotically Differentiated Human Neurons against Ethanol- and Human Immunodeficiency Virus Type 1-Induced Oxidative Stress In Vitro

2006 ◽  
Vol 81 (3) ◽  
pp. 1492-1501 ◽  
Author(s):  
Edward Acheampong ◽  
Zahida Parveen ◽  
Aschalew Mengistu ◽  
Noel Ngoubilly ◽  
Brian Wigdahl ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
The Brain ◽  
Hiv 1

ABSTRACT The majority of human immunodeficiency virus type 1 (HIV-1)-infected individuals are either alcoholics or prone to alcoholism. Upon ingestion, alcohol is easily distributed into the various compartments of the body, particularly the brain, by crossing through the blood-brain barrier. Both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In our studies, postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence of physiological concentrations of alcohol for 72 h. Parallel cultures were pretreated with statins (atorvastatin and simvastatin) with the appropriate controls, i.e., postmitotically differentiated neurons cocultured with uninfected cells and similar cultures treated with alcohol. The oxidative stress responses in the presence/absence of alcohol in these cultures were determined by the production of the well-characterized oxidative stress markers, 8-isoprostane-F2-α, total nitrates as an indicator for various isoforms of nitric oxide synthase activity, and heat shock protein 70 (Hsp70). An in vitro culture of postmitotically differentiated neurons with HIV-1-infected monocytes or T cells as well as supernatants from these cells enhanced the release of 8-isoprostane-F2-α in the conditioned medium six- to sevenfold (monocytes) and four- to fivefold (T cells). It was also observed that coculturing of HIV-1-infected primary monocytes over a time period of 72 h significantly elevated the release of Hsp70 compared with that of uninfected controls. Cellular supernatants of HIV-1-infected monocytes or T cells slightly increased Hsp70 levels compared to neurons cultured with uninfected monocytes or T-cell supernatants (controls). Ethanol (EtOH) presence further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the coculture system when both infected cells and EtOH were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters, i.e., Hsp70, isoprostanes, and total nitrates from HIV-1-infected cells. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain.

scholarly journals Comparative clonal analysis of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines.

1992 ◽  
Vol 176 (6) ◽  
pp. 1531-1542 ◽  
Author(s):  
S A Hammond ◽  
R C Bollinger ◽  
P E Stanhope ◽  
T C Quinn ◽  
D Schwartz ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytolytic T Lymphocytes ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were confirmed in studies in which CD4+ lymphoblasts were exposed to recombinant gp120 and used as targets for gp120-specific CD4+ and CD8+ CTL clones. gp120-pulsed, noninfected targets were lysed in an antigen-specific fashion by CD4+ but not CD8+ CTL clones. Taken together, these observations demonstrate that in an in vitro HIV-1 infection, sufficient amounts of gp120 antigen are produced and shed by infected cells to enable uptake by cells that are not yet infected, resulting in the lysis of these noninfected cells by gp120-specific, CD4+ CTL.(ABSTRACT TRUNCATED AT 400 WORDS)

Imidazo[1,5-b]Pyridazine-D4T Conjugates: Synthesis and Anti-Human Immunodeficiency Virus Evaluation

1998 ◽  
Vol 9 (3) ◽  
pp. 205-223 ◽  
Author(s):  
M Renoud-Grappin ◽  
C Fossey ◽  
G Fontaine ◽  
D Ladurée ◽  
AM Aubertin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Clear Trend ◽  
The Third ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Rt Inhibitors ◽  
Anti Hiv ◽  
Hiv 1

In an attempt to combine the human immunodeficiency virus type 1 (HIV-D-inhibitory capacity of 2′,3 -dideoxy-2,3 -didehydronucleoside analogues [nucleoside reverse transcriptase (RT) inhibitors; NRTI] and non-nucleoside RT inhibitors (NNRTI), we have designed, synthesized and evaluated for their anti-HIV activity several heterodimers of the general formula [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine]. The synthesis of these heterodimers was conducted in three parts. The first part focused on the synthesis of the NRTI. The second part was devoted to the NNRTI and the NNRTI linked to appropriate spacers; [NNRTI]-NH-(CH2)n-NH2. In the third part, the condensation between the NRTI and the [NNRTI]-NH-(CH2)n-NH2 was performed. The in vitro inhibitory activities against HIV-1 of the [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine] heterodimers were found to be comparable to that of d4T (stavudine) in HIV-infected cells. Moreover, the heterodimers were endowed with anti-HlV-2 activity and with anti-nevirapine-resistant HIV-1 activity. None of the heterodimers proved markedly cytotoxic to CEM-SS or MT-4 cells. There was not a clear trend toward antiviral potency on lengthening the methylene spacer in the [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine] heterodimers.

scholarly journals Native oligodeoxynucleotides specifically active against human immunodeficiency virus type 1 in vitro: a G-quartet-driven effect?

1996 ◽  
Vol 40 (9) ◽  
pp. 2034-2038 ◽  
Author(s):  
L Tondelli ◽  
F P Colonna ◽  
A Garbesi ◽  
S Zanella ◽  
M E Marongiu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
De Novo ◽  
Active Structure ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

Among a series of unmodified phosphodiester (PO)-oligodeoxynucleotides (PO-ODNs) complementary to some of the human immunodeficiency virus type 1 (HIV-1) regulatory genes, several PO-ODN sequences complementary to the vpr gene (PO-ODNs-a-vpr, where a-vpr is the antisense vpr sequence) emerged as potent inhibitors (at concentrations of 0.8 to 3.3 microM) of HIV-1 multiplication in de novo infected MT-4 cells, while they showed no cytotoxicity for uninfected cells at concentrations up to 100 microM. Unlike phosphorothioate counterparts, PO-ODN-a-vpr sequences were not inhibitory to HIV-2 multiplication in de novo infected C8166 cells and neither prevented the fusion between chronically infected and bystander CD4+ cells nor inhibited the activity of the HIV-1 reverse transcriptase in enzyme assays. Moreover, they were not inhibitory to HIV-1 multiplication in chronically infected cells. Delayed addition experiments showed that PO-ODNs-a-vpr inhibit an event in the HIV-1 replication cycle following adsorption to the host cell, but preceding reverse transcription. Structure-activity relationship studies indicated that the antiviral activity of the test PO-ODN-a-vpr sequences is not related to an antisense mechanism but to the presence, within the active sequences, of contiguous guanine residues. Physical characterization of the test PO-ODNs suggested that the active structure is a tetramer stabilized by G quartets (i.e., four G residues connected by eight hydrogen bonds).

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