scholarly journals Effect of Mutations in Gag on Assembly of Immature Human Immunodeficiency Virus Type 1 Capsids in a Cell-Free System

Virology ◽  
2001 ◽  
Vol 279 (1) ◽  
pp. 257-270 ◽  
Author(s):  
Aalok R. Singh ◽  
Rebecca L. Hill ◽  
Jaisri R. Lingappa
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Free System ◽  
Cell Free System ◽  
Immunodeficiency Virus ◽  
A Cell

scholarly journals Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction

2021 ◽  
Author(s):  
Tucker Hansen ◽  
Jodie Baris ◽  
Min Zhao ◽  
Richard Sutton
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Protein ◽  
Mutant Type ◽  
Free System ◽  
Rev Response Element ◽  
Cell Free System ◽  
Immunodeficiency Virus

Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing NLUC-Rev or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ~0.85 when tested in 96-well format, and anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

scholarly journals Human Immunodeficiency Virus Type 1 Latency Model for High-Throughput Screening

2005 ◽  
Vol 49 (12) ◽  
pp. 5185-5188 ◽  
Author(s):  
Sofiya Micheva-Viteva ◽  
Annmarie L. Pacchia ◽  
Yacov Ron ◽  
Stuart W. Peltz ◽  
Joseph P. Dougherty
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Throughput ◽  
High Throughput Screening ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
A Cell ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.

scholarly journals Topotecan inhibits human immunodeficiency virus type 1 infection through a topoisomerase-independent mechanism in a cell line with altered topoisomerase I.

1997 ◽  
Vol 41 (5) ◽  
pp. 977-981 ◽  
Author(s):  
J L Zhang ◽  
P L Sharma ◽  
C J Li ◽  
B J Dezube ◽  
A B Pardee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Topoisomerase I ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
A Cell ◽  
Hiv 1

Topotecan (TPT), a known inhibitor of topoisomerase I, has previously been shown to inhibit the replication of several viruses. The mechanism of inhibition was proposed to be the inhibition of topoisomerase I. We report that TPT decreased replication of human immunodeficiency virus type 1 (HIV-1) in CPT-K5, a cell line with a topoisomerase I mutation. TPT inhibited production of HIV-1 RNA and p24 in CPT-K5 and wild-type cells equally effectively. The antiviral effects of TPT were observed not only in the topoisomerase-mutated CPT-K5 line but also in peripheral blood mononuclear cells (PBMC) acutely infected with clinical isolates and in OM10.1 cells latently infected with HIV and activated by tumor necrosis factor alpha. Little toxicity from TPT was noted in HIV-1-infected PBMC and in CPT-K5 and OM10.1 cells as measured by cell growth and proliferation assays. These observations suggest that TPT targets factors in virus replication other than cellular topoisomerase I and inhibits cytokine-mediated activation in latently infected cells by means other than cytotoxicity. These results suggest a potential for TPT and for other camptothecins in anti-HIV therapy alone and in combination with other antiretroviral drugs.

scholarly journals A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

1999 ◽  
Vol 73 (11) ◽  
pp. 8966-8974 ◽  
Author(s):  
Alexandra Trkola ◽  
Jamie Matthews ◽  
Cynthia Gordon ◽  
Tom Ketas ◽  
John P. Moore
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Neutralization Assay ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Cxcr4 Coreceptor ◽  
Hiv 1

ABSTRACT We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.

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