scholarly journals Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities

2016 ◽  
Vol 90 (12) ◽  
pp. 5657-5664 ◽  
Author(s):  
Ayna Alfadhli ◽  
Andrew Mack ◽  
Christopher Ritchie ◽  
Isabel Cylinder ◽  
Logan Harper ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Matrix Protein ◽  
Rna Binding ◽  
Mutant Proteins ◽  
Virus Particles ◽  
Amino Terminal ◽  
Virion Incorporation ◽  
Multivalent Binding ◽  
Env Protein ◽  
Hiv 1

ABSTRACTThe HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.IMPORTANCEThe HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

scholarly journals Analysis of HIV-1 Matrix-Envelope Cytoplasmic Tail Interactions

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Ayna Alfadhli ◽  
August O. Staubus ◽  
Philip R. Tedbury ◽  
Mariia Novikova ◽  
Eric O. Freed ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Binding ◽  
Culture Studies ◽  
Virus Particles ◽  
Cytoplasmic Tails ◽  
The Matrix ◽  
Direct Binding ◽  
Env Protein ◽  
Rna Ligands ◽  
Hiv 1

ABSTRACT The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incorporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough-mapping studies indicate that C-terminal CT helices are involved in MA binding and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers. IMPORTANCE The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interfering with MA trimerization and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.

scholarly journals Retroviral Env Glycoprotein Trafficking and Incorporation into Virions

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Tsutomu Murakami
Keyword(s):  
Infectious Virus ◽  
Structural Data ◽  
Host Factors ◽  
Structural Protein ◽  
Gag Protein ◽  
Virus Particles ◽  
Virion Incorporation ◽  
Actual Mechanism ◽  
Env Protein ◽  
Env Glycoprotein

Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

scholarly journals The Carboxy-Terminal Domain Is Essential for Stability and Not for Virion Incorporation of HIV-1 Vpr into Virus Particles

Virology ◽  
1995 ◽  
Vol 214 (2) ◽  
pp. 647-652 ◽  
Author(s):  
S. MAHALINGAM ◽  
MAMATA PATEL ◽  
R.G. COLLMAN ◽  
A. SRINIVASAN
Keyword(s):  
Virus Particles ◽  
Terminal Domain ◽  
Virion Incorporation ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Hiv 1

scholarly journals Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly

2015 ◽  
Vol 89 (21) ◽  
pp. 10832-10840 ◽  
Author(s):  
Luca Sardo ◽  
Steven C. Hatch ◽  
Jianbo Chen ◽  
Olga Nikolaitchik ◽  
Ryan C. Burdick ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Light Exposure ◽  
Virus Assembly ◽  
Rna Packaging ◽  
Stem Loop ◽  
Hiv 1

ABSTRACTTo increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions.IMPORTANCENascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

scholarly journals Association of Potent Human Antiviral Cytidine Deaminases with 7SL RNA and Viral RNP in HIV-1 Virions

2010 ◽  
Vol 84 (24) ◽  
pp. 12903-12913 ◽  
Author(s):  
Wenyan Zhang ◽  
Juan Du ◽  
Kevin Yu ◽  
Tao Wang ◽  
Xiong Yong ◽  
...  
Keyword(s):  
Rna Binding ◽  
Signal Recognition Particle ◽  
Viral Inhibition ◽  
Cytidine Deaminases ◽  
Amino Terminal ◽  
7Sl Rna ◽  
Rnp Complex ◽  
Rna Interaction ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT 7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{3}^{Lys}\) \end{document} . Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.

scholarly journals Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly

2005 ◽  
Vol 79 (22) ◽  
pp. 13839-13847 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Matrix Protein ◽  
Particle Production ◽  
Rna Binding ◽  
Immunodeficiency Virus ◽  
The Matrix ◽  
Hiv 1

ABSTRACT RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.

scholarly journals Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

2012 ◽  
Vol 288 (1) ◽  
pp. 666-676 ◽  
Author(s):  
Ayna Alfadhli ◽  
Henry McNett ◽  
Jacob Eccles ◽  
Seyram Tsagli ◽  
Colleen Noviello ◽  
...  
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Matrix Protein ◽  
Rna Binding ◽  
Small Molecule Ligands ◽  
Hiv 1

scholarly journals Multiple Charged and Aromatic Residues in CCR5 Amino-terminal Domain Are Involved in High Affinity Binding of Both Chemokines and HIV-1 Env Protein

1999 ◽  
Vol 274 (49) ◽  
pp. 34719-34727 ◽  
Author(s):  
Cédric Blanpain ◽  
Benjamin J. Doranz ◽  
Jalal Vakili ◽  
Joseph Rucker ◽  
Cédric Govaerts ◽  
...  
Keyword(s):  
Affinity Binding ◽  
High Affinity Binding ◽  
Aromatic Residues ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Env Protein ◽  
Amino Terminal Domain ◽  
Hiv 1

scholarly journals Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites

1998 ◽  
Vol 72 (4) ◽  
pp. 2846-2854 ◽  
Author(s):  
Klaus Wiegers ◽  
Gabriel Rutter ◽  
Hubert Kottler ◽  
Uwe Tessmer ◽  
Heinz Hohenberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Matrix Protein ◽  
Rna Binding ◽  
Point Mutations ◽  
Dense Layer ◽  
Gag Polyprotein ◽  
C Terminus ◽  
Capsid Shell ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

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