The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (0NCp7), 1 (NCp7) or 2 (2NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, 2NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas 0NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.

Download Full-text

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the gag polyprotein

Journal of Molecular Biology ◽

10.1006/jmbi.2000.3960 ◽

2000 ◽

Vol 301 (5) ◽

pp. 1315

Author(s):

Amanda Zeffman ◽

Stuart Hassard ◽

Gabriele Varani ◽

Andrew Lever

Keyword(s):

Packaging Signal ◽

Stem Loop ◽

Gag Polyprotein ◽

Hiv 1

Download Full-text

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the gag polyprotein 1 1Edited by I. Tinoco

Journal of Molecular Biology ◽

10.1006/jmbi.2000.3611 ◽

2000 ◽

Vol 297 (4) ◽

pp. 877-893 ◽

Cited By ~ 49

Author(s):

Amanda Zeffman ◽

Stuart Hassard ◽

Gabriele Varani ◽

Andrew Lever

Keyword(s):

Packaging Signal ◽

Stem Loop ◽

Gag Polyprotein ◽

Hiv 1

Download Full-text

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation

Viruses ◽

10.3390/v13112312 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2312

Author(s):

Sébastien Lyonnais ◽

S. Kashif Sadiq ◽

Cristina Lorca-Oró ◽

Laure Dufau ◽

Sara Nieto-Marquez ◽

...

Keyword(s):

Self Assembly ◽

Rna Binding ◽

Weak Interactions ◽

Biological Functions ◽

Viral Protease ◽

Virus Maturation ◽

Sequential Processing ◽

Rnp Granules ◽

Crowded Environments ◽

Hiv 1

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

Download Full-text

ANTIVIRAL ACTIVITY OF STERIC-BLOCK OLIGONUCLEOTIDES TARGETING THE HIV-1 TRANS-ACTIVATION RESPONSE AND PACKAGING SIGNAL STEM-LOOP RNAS

Nucleosides Nucleotides & Nucleic Acids ◽

10.1081/ncn-200059813 ◽

2005 ◽

Vol 24 (5-7) ◽

pp. 393-396 ◽

Cited By ~ 7

Author(s):

Douglas Brown ◽

Andrey A. Arzumanov ◽

John J. Turner ◽

Dmitry A. Stetsenko ◽

Andrew M. L. Lever ◽

...

Keyword(s):

Antiviral Activity ◽

Packaging Signal ◽

Stem Loop ◽

Activation Response ◽

Hiv 1

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.01146-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 10832-10840 ◽

Cited By ~ 25

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.

Download Full-text

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

Viruses ◽

10.3390/v12040394 ◽

2020 ◽

Vol 12 (4) ◽

pp. 394 ◽

Cited By ~ 3

Author(s):

Tomas Kroupa ◽

Siddhartha A. K. Datta ◽

Alan Rein

Keyword(s):

Salt Concentration ◽

Electrostatic Interactions ◽

Rna Binding ◽

High Specificity ◽

Viral Rna ◽

Gag Protein ◽

Gag Polyprotein ◽

The Individual ◽

Hiv 1

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

Download Full-text

Basic Residues of the Retroviral Nucleocapsid Play Different Roles in Gag-Gag and Gag-Ψ RNA Interactions

Journal of Virology ◽

10.1128/jvi.78.16.8486-8495.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8486-8495 ◽

Cited By ~ 23

Author(s):

Eun-Gyung Lee ◽

Maxine L. Linial

Keyword(s):

Rna Binding ◽

Tertiary Structure ◽

Packaging Signal ◽

Gag Polyprotein ◽

Complex Interactions ◽

Charged Amino Acids ◽

C Terminus ◽

Gag Assembly ◽

Positively Charged

ABSTRACT The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (Ψ)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored Ψ RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for Ψ RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.

Download Full-text

Stem-loop SL4 of the HIV-1 Ψ RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition

Journal of Molecular Biology ◽

10.1006/jmbi.2000.5182 ◽

2001 ◽

Vol 314 (5) ◽

pp. 961-970 ◽

Cited By ~ 52

Author(s):

Gaya K. Amarasinghe ◽

Jing Zhou ◽

Matthew Miskimon ◽

Kalola J. Chancellor ◽

Jasmine A. McDonald ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Structural Studies ◽

Rna Packaging ◽

Packaging Signal ◽

Stem Loop ◽

Genome Recognition ◽

Rna Packaging Signal ◽

Hiv 1

Download Full-text

HIV-1 Packaging Visualised by In-Gel SHAPE

Viruses ◽

10.3390/v13122389 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2389

Author(s):

Aaron R. D’Souza ◽

Dhivya Jayaraman ◽

Ziqi Long ◽

Jingwei Zeng ◽

Liam J. Prestwood ◽

...

Keyword(s):

Rna Structure ◽

Structural Changes ◽

Structural Protein ◽

Packaging Signal ◽

Packaging Process ◽

Gag Polyprotein ◽

Rna Conformation ◽

Shape Selective ◽

Insight Into ◽

Hiv 1

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE (selective 2’OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

Download Full-text