Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Sun, Mengya; Grigsby, Iwen F.; Gorelick, Robert J.; Mansky, Louis M.; Musier‐Forsyth, Karin

doi:10.1128/jvi.02151-13

Cited by 25 publications

(39 citation statements)

References 60 publications

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“…The failure of IP6 to stimulate RSV Gag chaperone function likely is due to the weak binding of the MA domain to NA rather than to lack of MA-IP interaction based on our finding that IP6 competes with RNA for binding to MA. Thus, similar to other retroviral MA domains (20,56,57), RSV MA is capable of binding IPs, but unlike the case in HIV-1, this binding does not stimulate chaperone function because MA is so weakly bound to NA in the first place. Furthermore, finding that the RSV Gag-NA interaction was not disrupted by IP6 in vitro suggests that Gag is so tightly bound to NA via the NC domain that IP6 is not an effective competitor.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Rye-McCurdy

Nadaraia-Hoke

Gudleski-O'Regan

et al. 2014

J Virol

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Host cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNALys3 as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNA Trp . The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNA Lys3 to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNA Trp to a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (Gag⌬p6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 Gag⌬p6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 Gag⌬p6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function. IMPORTANCEMounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstrated in vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperone in vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.

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Section: Discussionmentioning

confidence: 99%

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Rye-McCurdy

Nadaraia-Hoke

Gudleski-O'Regan

et al. 2014

J Virol

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“…The N-terminal domain of Gag (matrix [MA]) facilitates both Gag-membrane and Gag-RNA interactions (10). The capsid (CA) domain is primarily responsible for Gag-Gag interactions, and the C-terminal region encodes the nucleocapsid (NC) domain, which is particularly crucial for retroviral RNA packaging and stabilizing CA-CA interactions (11,12).…”

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confidence: 99%

Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles

Martin

Cao

Maldonado

et al. 2016

J Virol

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The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of viruslike particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thinsection transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta-and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta-and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumaviruslike particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. IMPORTANCEComparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway. The Gag polyprotein is the primary retroviral structural protein responsible for orchestrating retrovirus assembly. HIV-1 Gag has arguably been the most extensively studied Gag polyprotein to date (1-7); however, distinct differences in the biology of retroviral Gag proteins have emphasized the importance of comparative analyses in order to gain further insights (8).The Gag polyprotein is functionally conserved across the two retroviral subfamilies (i.e., Orthoretrovirinae and Spumaretrovirinae). While the six genera in the Orthoretrovirinae subfamily (Alpha-, Beta-, Delta-, Epsilon-, Gamma-, and Lentivirus) an...

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“…In MLV, the virally encoded gag polyprotein undergoes proteolytic cleavage into three component proteins: matrix protein (MA), capsid (CA), and nucleocapsid (NC) (D'Souza et al, 2001). Of these, NC participates in the selective packaging of two copies of the unspliced full-length genome through its interaction with the viral RNA psi (Ψ) sequence (Sun et al, 2014). The Ψ site is located within the 5'-UTR of the viral genome and contains four stem loop structures, each of which participates in encapsidation, dimerization and transportation.…”

Section: Introductionmentioning

confidence: 99%

Identification of Porcine Endogenous Retrovirus (PERV) packaging sequence and development of PERV packaging viral vector system

et al. 2015

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Studies of the retroviruses have focused on the specific interaction of the nucleocapsid protein with a packaging signal in the viral RNA as important for this selectivity, but the packaging signal in porcine endogenous retrovirus (PERV) has not been defined. Herein, we identified and analyzed this packaging signal in PERV and found hairpin structures with conserved tetranucleotides in their loops and nucleocapsid recognition sequences; both of which are key elements in the viral packaging signal of MLV. We evaluated packaging efficiency of sequence variants isolated from viral and proviral integrated genomes. All viral packaging sequences (Ψ) were identical, while five distinct packaging sequences were identified from proviral sources. One proviral sequence (Ψ1) was identical to that of the viral Ψ and had the highest packaging efficiency. Three variants (Ψ2, Ψ3, Ψ4) maintained key elements of the viral packaging signal, but had nucleotide replacements and consequently demonstrated reduced packaging efficiency. Despite of the same overall hairpin structure, the proviral variant (Ψ5) had only one GACG sequence in the hairpin loop and showed the lowest packaging efficiency other than ∆Ψ, in which the essential packaging sequence was removed. This result, thus, defined the packaging sequences in PERV and emphasized the importance of nucleotide sequence and RNA structure in the determination of packaging efficiency. In addition, we demonstrate efficient infection and gene expression from the PERV based viral vector, which may serve as a novel alternative to current retroviral expression systems.

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Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Cited by 25 publications

References 60 publications

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles

Identification of Porcine Endogenous Retrovirus (PERV) packaging sequence and development of PERV packaging viral vector system

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