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 Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

1998 ◽  
Vol 72 (4) ◽  
pp. 2935-2944 ◽  
Author(s):  
Sarah L. Thomas ◽  
Martin Oft ◽  
Herbert Jaksche ◽  
Georg Casari ◽  
Peter Heger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Protein ◽  
Surface Patch ◽  
Target Sequence ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viraltrans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form atrans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.

scholarly journals Human Immunodeficiency Virus Type 1 Tat Induces Apoptosis and Increases Sensitivity to Apoptotic Signals by Up-Regulating FLICE/Caspase-8

1999 ◽  
Vol 73 (3) ◽  
pp. 1956-1963 ◽  
Author(s):  
Steven R. Bartz ◽  
Michael Emerman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Protein ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Immunodeficiency Virus ◽  
Induction Of Apoptosis ◽  
Hiv 1

ABSTRACT Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the envgene (HIVΔenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.

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