scholarly journals Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative

2004 ◽  
Vol 78 (19) ◽  
pp. 10803-10813 ◽  
Author(s):  
Barbara Müller ◽  
Jessica Daecke ◽  
Oliver T. Fackler ◽  
Matthias T. Dittmar ◽  
Hanswalter Zentgraf ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Live Cells ◽  
Structural Protein ◽  
Initial Finding ◽  
Particle Assembly ◽  
Immunodeficiency Virus

ABSTRACT The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.

scholarly journals Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells

2006 ◽  
Vol 80 (17) ◽  
pp. 8796-8806 ◽  
Author(s):  
Candace Y. Gomez ◽  
Thomas J. Hope
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Therapy-Naive Individuals Residing in Sorong, West Papua

2016 ◽  
Vol 32 (8) ◽  
pp. 812-817 ◽  
Author(s):  
Adiana Mutamsari Witaningrum ◽  
Tomohiro Kotaki ◽  
Siti Qamariyah Khairunisa ◽  
Muhammad Qushai Yunifiar M ◽  
Dwi Wahyu Indriati ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genotypic Characterization ◽  
Immunodeficiency Virus ◽  
West Papua

scholarly journals Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

2005 ◽  
Vol 79 (23) ◽  
pp. 14498-14506 ◽  
Author(s):  
Ayna Alfadhli ◽  
Tenzin Choesang Dhenub ◽  
Amelia Still ◽  
Eric Barklis
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protein Assembly ◽  
Particle Assembly ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Terminal Domains

ABSTRACT The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

scholarly journals Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins

2008 ◽  
Vol 82 (21) ◽  
pp. 10864-10872 ◽  
Author(s):  
Angsana Phuphuakrat ◽  
Romchat Kraiwong ◽  
Chompunuch Boonarkart ◽  
Darat Lauhakirti ◽  
Tun-Hou Lee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protein Expression ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Structural Protein ◽  
Double Stranded Rna ◽  
Adenosine Deaminases ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression.

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