scholarly journals HIV-1 integrase binding to genomic RNA 5′-UTR induces local structural changes in vitro and in virio

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Shuohui Liu ◽  
Pratibha C. Koneru ◽  
Wen Li ◽  
Chathuri Pathirage ◽  
Alan N. Engelman ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Structural Changes ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Virus Particles ◽  
Structure Changes ◽  
Apical Loop ◽  
Viral Genomic Rna ◽  
Hiv 1

Abstract Background During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. HIV-1 integrase (IN) binds the viral genomic RNA (gRNA) and impairment of IN-gRNA binding leads to mis-localization of the nucleocapsid protein (NC)-condensed viral ribonucleoprotein complex outside the capsid core. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; however, the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood. Results Using crosslinking-coupled selective 2′-hydroxyl acylation analyzed by primer extension (XL-SHAPE), we characterized the interaction of IN and NC with the HIV-1 gRNA 5′-untranslated region (5′-UTR). NC preferentially bound to the packaging signal (Psi) and a UG-rich region in U5, irrespective of the presence of IN. IN alone also bound to Psi but pre-incubation with NC largely abolished this interaction. In contrast, IN specifically bound to and affected the nucleotide (nt) dynamics of the apical loop of the transactivation response element (TAR) and the polyA hairpin even in the presence of NC. SHAPE probing of the 5′-UTR RNA in virions produced from allosteric IN inhibitor (ALLINI)-treated cells revealed that while the global secondary structure of the 5′-UTR remained unaltered, the inhibitor treatment induced local reactivity differences, including changes in the apical loop of TAR that are consistent with the in vitro results. Conclusions Overall, the binding interactions of NC and IN with the 5′-UTR are largely orthogonal in vitro. This study, together with previous probing experiments, suggests that IN and NC binding in vitro and in virio lead to only local structural changes in the regions of the 5′-UTR probed here. Accordingly, disruption of IN-gRNA binding by ALLINI treatment results in local rather than global secondary structure changes of the 5′-UTR in eccentric virus particles. Graphical Abstract

scholarly journals Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging

2006 ◽  
Vol 87 (10) ◽  
pp. 3039-3044 ◽  
Author(s):  
Jane S. Greatorex ◽  
Elizabeth A. Palmer ◽  
Roger J. Pomerantz ◽  
John A. Dangerfield ◽  
Andrew M. L. Lever
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Export ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Virus Particles ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Viral Genomic Rna ◽  
Human Immunodeficiency Virus 1

An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev–loop A interaction supplementary to or preceding that of the Rev–RRE interaction. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev–loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30 %. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98 % conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.

scholarly journals Efficient support of virus-like particle assembly by the HIV-1 packaging signal

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mauricio Comas-Garcia ◽  
Tomas Kroupa ◽  
Siddhartha AK Datta ◽  
Demetria P Harvin ◽  
Wei-Shau Hu ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Structural Element ◽  
Particle Assembly ◽  
Genomic Rnas ◽  
Gag Protein ◽  
Virus Particles ◽  
Virus Like Particle ◽  
Hiv 1

The principal structural component of a retrovirus particle is the Gag protein. Retroviral genomic RNAs contain a ‘packaging signal’ (‘Ψ') and are packaged in virus particles with very high selectivity. However, if no genomic RNA is present, Gag assembles into particles containing cellular mRNA molecules. The mechanism by which genomic RNA is normally selected during virus assembly is not understood. We previously reported (<xref ref-type="bibr" rid="bib9">Comas-Garcia et al., 2017</xref>) that at physiological ionic strength, recombinant HIV-1 Gag binds with similar affinities to RNAs with or without Ψ, and proposed that genomic RNA is selectively packaged because binding to Ψ initiates particle assembly more efficiently than other RNAs. We now present data directly supporting this hypothesis. We also show that one or more short stretches of unpaired G residues are important elements of Ψ; Ψ may not be localized to a single structural element, but is probably distributed over >100 bases.

scholarly journals Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2451
Author(s):  
Ivana Křížová ◽  
Alžběta Dostálková ◽  
Edison Castro ◽  
Jan Prchal ◽  
Romana Hadravová ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Biological Activities ◽  
Reverse Transcriptase Activity ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Fullerene Derivatives ◽  
Viral Genomic Rna ◽  
Anti Hiv ◽  
Hiv 1

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives’ action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription—without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives’ oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

scholarly journals Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection

RNA ◽  
2001 ◽  
Vol 7 (4) ◽  
pp. 576-584 ◽  
Author(s):  
PRAKASH PUROHIT ◽  
STEFAN DUPONT ◽  
MARIO STEVENSON ◽  
MICHAEL R. GREEN
Keyword(s):  
Matrix Protein ◽  
Specific Interaction ◽  
Genetic Selection ◽  
Genomic Rna ◽  
Viral Genomic Rna ◽  
Hiv 1

scholarly journals Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rebecca S. Brown ◽  
Dimitrios G. Anastasakis ◽  
Markus Hafner ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Binding Sites ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Genome Recognition ◽  
Infected Cells

Abstract The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

scholarly journals In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e54384 ◽  
Author(s):  
Shreyas S. Athavale ◽  
J. Jared Gossett ◽  
Jessica C. Bowman ◽  
Nicholas V. Hud ◽  
Loren Dean Williams ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Genomic Rna ◽  
Satellite Tobacco Mosaic Virus

scholarly journals Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

RNA ◽  
2013 ◽  
Vol 19 (8) ◽  
pp. 1078-1088 ◽  
Author(s):  
J. A. Webb ◽  
C. P. Jones ◽  
L. J. Parent ◽  
I. Rouzina ◽  
K. Musier-Forsyth
Keyword(s):  
Genomic Rna ◽  
Rna Packaging ◽  
Binding Interactions ◽  
Viral Genomic Rna ◽  
Hiv 1

scholarly journals Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

2005 ◽  
Vol 79 (22) ◽  
pp. 13848-13855 ◽  
Author(s):  
Ping-Kun Hsieh ◽  
Shin C. Chang ◽  
Chu-Chun Huang ◽  
Ting-Ting Lee ◽  
Ching-Wen Hsiao ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Culture Medium ◽  
Binding Activity ◽  
Viral Rna ◽  
Structural Proteins ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Virus Like Particles ◽  
N Protein ◽  
Viral Genomic Rna

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm3. Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3′ noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

scholarly journals Role of RNA in Facilitating Gag/Gag-Pol Interaction

2002 ◽  
Vol 76 (8) ◽  
pp. 4131-4137 ◽  
Author(s):  
Ahmad Khorchid ◽  
Rabih Halwani ◽  
Mark A. Wainberg ◽  
Lawrence Kleiman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Membrane Bound ◽  
Bulk Membrane ◽  
Triton X ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT We have examined the influence of RNA upon the interaction of Gag-Pol with Gag during human immunodeficiency virus type 1 (HIV-1) assembly. COS7 cells were transfected with protease-negative HIV-1 proviral DNA, and Gag/Gag-Pol complexes were detected by coimmunoprecipitation with anti-integrase. In COS7 cells, Gag/Gag-Pol is found almost entirely in pelletable, membrane-bound complexes. Exposure of cells to 1% Triton X-100 releases Gag/Gag-Pol from bulk membrane, but the complexes remain pelletable. The role of RNA in facilitating the interaction between Gag and Gag-Pol was examined in these bulk membrane-free, pelletable complexes. The specific presence of viral genomic RNA is not required to maintain the Gag/Gag-Pol interaction, but some type of RNA is, since exposure to RNase destabilized the Gag/Gag-Pol complex. When present only in Gag, the nucleocapsid mutation R7R10K11S, which inhibits Gag binding to RNA, inhibits the formation of both Gag and Gag/Gag-Pol complexes. When present only in Gag-Pol, this mutation has no effect upon complex formation. This result indicates that Gag-Pol may not interact directly with RNA but rather requires RNA-facilitated Gag multimerization for its interaction with Gag.

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