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

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.

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Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients

Blood ◽

10.1182/blood.v80.8.2128.2128 ◽

1992 ◽

Vol 80 (8) ◽

pp. 2128-2135 ◽

Cited By ~ 74

Author(s):

MP Busch ◽

TH Lee ◽

J Heitman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Blood Components ◽

Route Of Transmission ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Donor Lymphocytes

Abstract Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a “latently” infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.

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The Interaction of Vpr with Uracil DNA Glycosylase Modulates the Human Immunodeficiency Virus Type 1 In Vivo Mutation Rate

Journal of Virology ◽

10.1128/jvi.74.15.7039-7047.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 7039-7047 ◽

Cited By ~ 126

Author(s):

Louis M. Mansky ◽

Sandra Preveral ◽

Luc Selig ◽

Richard Benarous ◽

Serge Benichou

Keyword(s):

Human Immunodeficiency Virus ◽

Mutation Rate ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dna Glycosylase ◽

Uracil Dna Glycosylase ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.

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In vivo characteristics of human immunodeficiency virus type 1 intersubtype recombination: determination of hot spots and correlation with sequence similarity

Journal of General Virology ◽

10.1099/vir.0.19180-0 ◽

2003 ◽

Vol 84 (10) ◽

pp. 2715-2722 ◽

Cited By ~ 51

Author(s):

Gkikas Magiorkinis ◽

Dimitrios Paraskevis ◽

Anne-Mieke Vandamme ◽

Emmanouil Magiorkinis ◽

Vana Sypsa ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Recombination Frequency ◽

Sequence Similarity ◽

Virus Species ◽

Immunodeficiency Virus ◽

Hiv 1

Recombination plays a pivotal role in the evolutionary process of many different virus species, including retroviruses. Analysis of all human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants revealed that they are more complex than described initially. Recombination frequency is higher within certain genomic regions, such as partial reverse transcriptase (RT), vif/vpr, the first exons of tat/rev, vpu and gp41. A direct correlation was observed between recombination frequency and sequence similarity across the HIV-1 genome, indicating that sufficient sequence similarity is required upstream of the recombination breakpoint. This finding suggests that recombination in vivo may occur preferentially during reverse transcription through the strand displacement-assimilation model rather than the copy-choice model.

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Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.01213-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 9875-9889 ◽

Cited By ~ 10

Author(s):

Elodie Beaumont ◽

Daniela Vendrame ◽

Bernard Verrier ◽

Emmanuelle Roch ◽

François Biron ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Matrix Protein ◽

Immunodeficiency Virus ◽

The Matrix ◽

Hiv 1

ABSTRACT Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.

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Induction of Primary Virus-Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Antibodies in Small Animals by Using an Alphavirus-Derived In Vivo Expression System

Journal of Virology ◽

10.1128/jvi.77.5.3119-3130.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3119-3130 ◽

Cited By ~ 40

Author(s):

Ming Dong ◽

Peng Fei Zhang ◽

Franziska Grieder ◽

James Lee ◽

Govindaraj Krishnamurthy ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralizing Antibodies ◽

Neutralizing Activity ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Primary Virus

ABSTRACT We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160ΔCT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160ΔCT was released by cells in the form of membrane-bound vesicles. gp160ΔCT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virus-neutralizing activity by vaccination may be realized.

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In Vivo gp41 Antibodies Targeting the 2F5 Monoclonal Antibody Epitope Mediate Human Immunodeficiency Virus Type 1 Neutralization Breadth

Journal of Virology ◽

10.1128/jvi.02631-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3617-3625 ◽

Cited By ~ 82

Author(s):

Xiaoying Shen ◽

Robert J. Parks ◽

David C. Montefiori ◽

Jennifer L. Kirchherr ◽

Brandon F. Keele ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Vaccine Development ◽

Natural Infection ◽

Human Monoclonal Antibodies ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10, both targeting the highly conserved human immunodeficiency virus type 1 (HIV-1) envelope membrane proximal external region (MPER), are among the MAbs with the broadest heterologous neutralizing activity and are of considerable interest for HIV-1 vaccine development. We have identified serum antibodies from an HIV-infected subject that both were broadly neutralizing and specifically targeted MPER epitopes that overlap the 2F5 epitope. These MPER-specific antibodies were made 15 to 20 months following transmission and concomitantly with the development of autoantibodies. Our findings suggest that multiple events (i.e., genetic predisposition and HIV-1 immune dysregulation) may be required for induction of broadly reactive gp41 MPER antibodies in natural infection.

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A functional genetic approach suggests a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Tat protein and HIV-1 TAR RNA in vivo

Journal of General Virology ◽

10.1099/vir.0.18645-0 ◽

2003 ◽

Vol 84 (3) ◽

pp. 603-606 ◽

Cited By ~ 5

Author(s):

Lars H. Lund ◽

Britta Wahren ◽

Mariano A. Garcia-Blanco

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Tat Protein ◽

Cyclin T1 ◽

Immunodeficiency Virus ◽

Tar Rna ◽

Hiv 1

Human immunodeficiency virus type 1 (HIV-1) Tat and human Cyclin T1 form a complex and together recognize the viral TAR RNA element with specificity. Using HIV-1/equine infectious anaemia virus TAR chimeras, we show that in addition to the well-characterized interaction with the bulge, Tat recognizes the distal stem and the loop of TAR. These data support previously proposed, but unproven, molecular models.

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Separation of Human Immunodeficiency Virus Type 1 Replication from nef-Mediated Pathogenesis in the Human Thymus

Journal of Virology ◽

10.1128/jvi.75.8.3916-3924.2001 ◽

2001 ◽

Vol 75 (8) ◽

pp. 3916-3924 ◽

Cited By ~ 21

Author(s):

Karen M. Duus ◽

Eric D. Miller ◽

Jonathan A. Smith ◽

Grigoriy I. Kovalev ◽

Lishan Su

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

In Vivo Model ◽

Immunodeficiency Virus ◽

Pathogenic Activity ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is frequently attenuated after long-term culture in vitro. The attenuation process probably involves mutations of functions required for replication and pathogenicity in vivo. Analysis of attenuated HIV-1 for replication and pathogenicity in vivo will help to define these functions. In this study, we examined the pathogenicity of an attenuated HIV-1 isolate in a laboratory worker accidentally exposed to a laboratory-adapted HIV-1 isolate. Using heterochimeric SCID-hu Thy/Liv mice as an in vivo model, we previously defined HIV-1 env determinants (HXB/LW) that reverted to replicate in vivo (L. Su, H. Kaneshima, M. L. Bonyhadi, R. Lee, J. Auten, A. Wolf, B. Du, L. Rabin, B. H. Hahn, E. Terwilliger, and J. M. McCune, Virology 227:46–52, 1997). Here we further demonstrate that HIV-1 replication in vivo can be separated from its pathogenic activity, in that the HXB/LW virus replicated to high levels in SCID-hu Thy/Liv mice, with no significant thymocyte depletion. Restoration of the nef gene in the recombinant HXB/LW genome restored its pathogenic activity, with no significant effect on HIV-1 replication in the thymus. Our results suggest that in vitro-attenuated HIV-1 lacks determinants for pathogenicity as well as for replication in vivo. Our data indicate that (i) the replication defect can be recovered in vivo by mutations in the envgene, without an associated pathogenic phenotype, and (ii)nef can function in the HXB/LW clone as a pathogenic factor that does not enhance HIV-1 replication in the thymus. Furthermore, the HXB/LW virus may be used to study mechanisms of HIV-1nef-mediated pathogenesis in vivo.

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Contrasting Use of CCR5 Structural Determinants by R5 and R5X4 Variants within a Human Immunodeficiency Virus Type 1 Primary Isolate Quasispecies

Journal of Virology ◽

10.1128/jvi.77.22.12057-12066.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 12057-12066 ◽

Cited By ~ 9

Author(s):

Yanjie Yi ◽

Anjali Singh ◽

Farida Shaheen ◽

Andrew Louden ◽

ChuHee Lee ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Terminal Region ◽

Primary Isolate ◽

Structural Basis ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6PI quasispecies. R5 variants of 89.6PI could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6PI were highly dependent on the CCR5 N terminus. Similarly, R5 89.6PI variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6PI Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6PI was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.

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Increased Neutralization Sensitivity and Reduced Replicative Capacity of Human Immunodeficiency Virus Type 1 after Short-Term In Vivo or In Vitro Passage through Chimpanzees

Journal of Virology ◽

10.1128/jvi.74.17.7699-7707.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7699-7707 ◽

Cited By ~ 12

Author(s):

Tim Beaumont ◽

Silvia Broersen ◽

Ad van Nuenen ◽

Han G. Huisman ◽

Ana-Maria de Roda Husman ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Short Term ◽

Neutralization Sensitivity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.

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