scholarly journals Human Immunodeficiency Virus Env-Independent Infection of Human CD4− Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 10994-11000 ◽  
Author(s):  
Shen Pang ◽  
Duan Yu ◽  
Dong Sung An ◽  
Gayle C. Baldwin ◽  
Yiming Xie ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Alternative Pathway ◽  
Neutralizing Antibody ◽  
Green Fluorescent Protein Gene ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT CD4− epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4− human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4− cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.

scholarly journals Genetic Recombination between Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2, Two Distinct Human Lentiviruses

2007 ◽  
Vol 82 (4) ◽  
pp. 1923-1933 ◽  
Author(s):  
Kazushi Motomura ◽  
Jianbo Chen ◽  
Wei-Shau Hu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Genetic Recombination ◽  
Green Fluorescent Protein Gene ◽  
Low Frequencies ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are genetically distinct viruses that each can cause AIDS. Approximately 1 million people are infected with both HIV-1 and HIV-2. Additionally, these two viruses use the same receptor and coreceptors and can therefore infect the same target cell populations. To explore potential genetic interactions, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect the copackaging of HIV-1 and HIV-2 RNAs. The GFP-positive (GFP+) phenotype was detected in approximately 0.2% of the infection events, which was 35-fold lower than the intrasubtype HIV-1 rates. We isolated and characterized 54 GFP+ single-cell clones and determined that all of them contained proviruses with reconstituted gfp. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns, including those that had crossovers only in gfp. The most common hybrid genomes had heterologous long terminal repeats. Although infrequent, crossovers in the viral sequences were also identified. Taken together, our study demonstrates that HIV-1 and HIV-2 can recombine, albeit at low frequencies. These observations indicate that multiple factors are likely to restrict the generation of viable hybrid HIV-1 and HIV-2 viruses. However, considering the large coinfected human population and the high viral load in patients, these rare events could provide the basis for the generation of novel human immunodeficiency viruses.

scholarly journals Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

2001 ◽  
Vol 45 (9) ◽  
pp. 2616-2622 ◽  
Author(s):  
Kristina Lindsten ◽  
Tat'ána Uhlı́ková ◽  
Jan Konvalinka ◽  
Maria G. Masucci ◽  
Nico P. Dantuma
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Autocatalytic Cleavage ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.

scholarly journals Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

2001 ◽  
Vol 75 (17) ◽  
pp. 7944-7955 ◽  
Author(s):  
Noriko Nakajima ◽  
Richard Lu ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Dna Recombination ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

scholarly journals A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans

2004 ◽  
Vol 85 (4) ◽  
pp. 911-919 ◽  
Author(s):  
Matilu Mwau ◽  
Inese Cebere ◽  
Julian Sutton ◽  
Priscilla Chikoti ◽  
Nicola Winstone ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Clinical Trials ◽  
Vaccinia Virus ◽  
Mononuclear Cells ◽  
Vaccine Development ◽  
Cell Mediated Immunity ◽  
Phase I Clinical Trials ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Hiv 1

The immunogenicities of candidate DNA- and modified vaccinia virus Ankara (MVA)-vectored human immunodeficiency virus (HIV) vaccines were evaluated on their own and in a prime–boost regimen in phase I clinical trials in healthy uninfected individuals in the United Kingdom. Given the current lack of approaches capable of inducing broad HIV-neutralizing antibodies, the pTHr.HIVA DNA and MVA.HIVA vaccines focus solely on the induction of cell-mediated immunity. The vaccines expressed a common immunogen, HIVA, which consists of consensus HIV-1 clade A Gag p24/p17 proteins fused to a string of clade A-derived epitopes recognized by cytotoxic T lymphocytes (CTLs). Volunteers' fresh peripheral blood mononuclear cells were tested for HIV-specific responses in a validated gamma interferon enzyme-linked immunospot (ELISPOT) assay using four overlapping peptide pools across the Gag domain and three pools of known CTL epitopes present in all of the HIVA protein. Both the DNA and the MVA vaccines alone and in a DNA prime–MVA boost combination were safe and induced HIV-specific responses in 14 out of 18, seven out of eight and eight out of nine volunteers, respectively. These results are very encouraging and justify further vaccine development.

scholarly journals Neutralizing Antibody Responses in Acute Human Immunodeficiency Virus Type 1 Subtype C Infection

2007 ◽  
Vol 81 (12) ◽  
pp. 6187-6196 ◽  
Author(s):  
E. S. Gray ◽  
P. L. Moore ◽  
I. A. Choge ◽  
J. M. Decker ◽  
F. Bibollet-Ruche ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Subtype C ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

scholarly journals Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses

2007 ◽  
Vol 82 (2) ◽  
pp. 638-651 ◽  
Author(s):  
Yun Li ◽  
Bradley Cleveland ◽  
Igor Klots ◽  
Bruce Travis ◽  
Barbra A. Richardson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Pronounced Increase ◽  
Enhanced Sensitivity ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.

scholarly journals Upregulation of c-Fos in Activated T Lymphoid and Monocytic Cells by Human Immunodeficiency Virus-1 Tat Protein

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1654-1664 ◽  
Author(s):  
Davide Gibellini ◽  
Antonella Caputo ◽  
Silvano Capitani ◽  
Michele La Placa ◽  
Giorgio Zauli
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Promoter Activity ◽  
Jurkat Cells ◽  
U937 Cells ◽  
Tat Protein ◽  
Fos Protein ◽  
Significant Difference ◽  
Immunodeficiency Virus ◽  
Hiv 1

Abstract The regulatory Tat protein of the human immunodeficiency virus type-1 (HIV-1) is essential for viral replication and also shows pleiotropic activities on various cell functions. To get further insights into the molecular mechanisms underlying the biological activity of Tat, we investigated the effect of endogenous and exogenous Tat protein on c-fos gene expression in T lymphoblastoid (Jurkat) and monocytic (U937) cell lines, as well as in primary peripheral blood mononuclear cells (PBMC). Transient cotransfection of tat cDNA in sense orientation (tat/S), together with a plasmid containing the c-fos promoter (FC3, from −711 to +42) in front of the bacterial chloramphenicol acetyltransferase (CAT) gene significantly enhanced CAT activity in Jurkat cells activated by the addition of 15% fetal calf serum (FCS) or 5 μg/mL phytohemagglutinin plus 10−7 mol/L phorbol myristate acetate (PMA) and U937 cells activated by 15% FCS or 10−7 mol/L PMA. This effect was specifically due to Tat, since Jurkat and U937 cells cotransfected either with tat cDNA in antisense orientation (tat/AS), tat carrying a mutation in the aminoacid cys22 - gly22 (tat 22/S) or with the backbone vector alone (pRPneo-SL3) did not show any significant difference in c-fos promoter activity as compared to cells transfected with FC3 plasmid alone. By using deletion mutants of the c-fos promoter, we found that the minimal DNA sequence required for Tat activity was located between nucleotides −404/−220 and that the serum responsive element (SRE, −317/−288), present within this region, was still responsive to Tat. A single point mutation in the SRE completely abrogated the responsiveness to tat/S. Exogenous recombinant Tat protein was also able to upregulate c-fos promoter activity in serum-activated Jurkat and U937 cells, as well as endogenous c-fos mRNA expression and c-Fos protein synthesis in both serum-activated cell lines and primary PBMC. c-Fos protein was shown essential for an optimal transactivation of the HIV-1 long terminal repeat (LTR) by Tat: incubation of Jurkat cells with antisense, but not sense, c-fos oligonucleotides significantly reduced either the Tat-enhanced expression of an LTR-CAT reporter construct or the levels of gag p24 in the culture supernatants of Jurkat cells and PBMC acutely infected with HIV-1. Our data suggest that the c-fos upregulation mediated by Tat might play a significant role in the control of viral gene transactivation.

scholarly journals A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120

2008 ◽  
Vol 82 (13) ◽  
pp. 6359-6368 ◽  
Author(s):  
Rena D. Astronomo ◽  
Hing-Ken Lee ◽  
Christopher N. Scanlan ◽  
Ralph Pantophlet ◽  
Cheng-Yuan Huang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Neutralizing Antibody ◽  
Humoral Immune ◽  
Aids Vaccine ◽  
Humoral Immune Responses ◽  
Copy Numbers ◽  
Immunodeficiency Virus ◽  
Broadly Neutralizing Antibody ◽  
Hiv 1

ABSTRACT The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man4) that corresponds to the D1 arm of Man9GlcNAc2 inhibits 2G12 binding to gp120 as efficiently as Man9GlcNAc2 itself, indicating the potential use of Man4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man4 up to a limit which is achieved at ∼10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man4)14 elicits significant serum Ab titers to Man4. However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1.

scholarly journals An Unrelated Monoclonal Antibody Neutralizes Human Immunodeficiency Virus Type 1 by Binding to an Artificial Epitope Engineered in a Functionally Neutral Region of the Viral Envelope Glycoproteins

2005 ◽  
Vol 79 (9) ◽  
pp. 5616-5624 ◽  
Author(s):  
Xinping Ren ◽  
Joseph Sodroski ◽  
Xinzhen Yang
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Envelope Glycoprotein ◽  
Virus Entry ◽  
Neutralizing Antibody ◽  
Viral Envelope ◽  
Epitope Tag ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Neutralizing antibodies often recognize regions of viral envelope glycoproteins that play a role in receptor binding or other aspects of virus entry. To address whether this is a necessary feature of a neutralizing antibody, we identified the V4 region of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) as a sequence that is tolerant of drastic change and thus appears to play a negligible role in envelope glycoprotein function. An artificial epitope tag was inserted into the V4 region without a significant effect on virus entry or neutralization by antibodies that recognize HIV-1 envelope glycoprotein sequences. An antibody directed against the artificial epitope tag was able to neutralize the modified, but not the wild-type, HIV-1. Thus, the specific target of a neutralizing antibody need not contribute functionally to the process of virus entry.

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