scholarly journals Zinc-binding properties of Junín virus nucleocapsid protein

2001 ◽  
Vol 82 (1) ◽  
pp. 121-128 ◽  
Author(s):  
M. Alejandra Tortorici ◽  
P. Daniel Ghiringhelli ◽  
Mario E. Lozano ◽  
César G. Albariño ◽  
Víctor Romanowski
Keyword(s):  
Nucleocapsid Protein ◽  
Zinc Binding ◽  
Dual Function ◽  
Genome Replication ◽  
Divalent Metal Ions ◽  
Junin Virus ◽  
N Protein ◽  
Carboxy Terminal ◽  
Junín Virus

The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

Polysulfonates Derived from Metal Thiolate Complexes as Inhibitors of HIV-1 and Various other Enveloped Viruses In Vitro

2002 ◽  
Vol 13 (3) ◽  
pp. 185-195 ◽  
Author(s):  
Donald E Bergstrom ◽  
Xiaoping Lin ◽  
Troy D Wood ◽  
Myriam Witvrouw ◽  
Satoru Ikeda ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Palladium Complex ◽  
Platinum Complex ◽  
Cadmium Complex ◽  
Junin Virus ◽  
Tacaribe Virus ◽  
Junín Virus ◽  
Hiv 1 ◽  
Hsv 1

Sodium 2-mercaptoethanesulfonate reacts with the metal ions Pd(II), Pt(II), Ag(I), Cd(II) and Zn(II) to yield complexes containing multiple anionic sulfonate sites. On the basis of spectroscopic and other analytical data the complexes were assigned the tentative molecular formulas: Pd6(SCH2CH2SO3Na)12, Ptn(SCH2CH2SO3Na)2n+2, Agn(SCH2CH2SO3Na)n, Na2Zn4(SCH2CH2SO3Na)10, and Na2Cd4(SCH2CH2SO3Na)10. The complexes displayed a variety of differences in activity towards DNA and RNA viruses. The platinum complex showed no measurable cytotoxicity and exhibited a spectrum of antiviral activity resembling that of dextran sulfate. It was active against HIV-1 and HIV-2, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine kinase-deficient HSV-1, human cytomegalovirus, vesicular stomatitis virus (VSV), influenza A virus, respiratory syncytial virus (RSV), Sindbis virus, Junin virus and Tacaribe virus. The palladium complex also showed no measurable cytotoxicity, but was completely inactive against most viruses, with one notable exception: both HIV-1 and HIV-2 were substantially inhibited by the palladium complex. The silver complex showed significantly less antiviral activity and greater cytotoxicity than the platinum complex but did show some selectivity against RSV. The zinc complex showed only modest activity against VSV, RSV, Junin virus, and Tacaribe virus, and like the silver compound was more cytotoxic than either the platinum or palladium complex. The cadmium complex was toxic to all of the cell lines used for in vitro evaluation of antiviral activity. Based on these results, the platinum and palladium compounds appear to be promising candidates for further studies, that is, as vaginal microbicides in the prevention of genital HIV and/or HSV transmission.

In vitro selection of Junin virus antigenic variants

1993 ◽  
Vol 128 (3-4) ◽  
pp. 389-394 ◽  
Author(s):  
Laura E. Alché ◽  
Celia E. Coto
Keyword(s):  
In Vitro Selection ◽  
Junin Virus ◽  
Junín Virus ◽  
Selection Of

scholarly journals Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein

2009 ◽  
Vol 83 (14) ◽  
pp. 7221-7234 ◽  
Author(s):  
Kelley R. Hurst ◽  
Cheri A. Koetzner ◽  
Paul S. Masters
Keyword(s):  
Nucleocapsid Protein ◽  
Fluorescent Protein ◽  
Hepatitis Virus ◽  
M Protein ◽  
N Protein ◽  
Terminal Domain ◽  
Domain 3 ◽  
Positive Strand Rna ◽  
Carboxy Terminal

ABSTRACT The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.

scholarly journals Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

2005 ◽  
Vol 12 (1) ◽  
pp. 114-121 ◽  
Author(s):  
Kang-Seuk Choi ◽  
Jin-Ju Nah ◽  
Young-Joon Ko ◽  
Shien-Young Kang ◽  
Kyoung-Jin Yoon ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peste Des Petits Ruminants ◽  
Serum Antibodies ◽  
N Protein ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Antigenic Domains ◽  
Carboxy Terminal

ABSTRACT Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.

scholarly journals In vitro infection of murine macrophages with Junin virus.

1982 ◽  
Vol 35 (1) ◽  
pp. 356-358 ◽  
Author(s):  
P H González ◽  
J S Lampuri ◽  
C E Coto ◽  
R P Laguens
Keyword(s):  
Junin Virus ◽  
Murine Macrophages ◽  
Junín Virus

scholarly journals Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

2016 ◽  
Vol 90 (9) ◽  
pp. 4357-4368 ◽  
Author(s):  
Lili Kuo ◽  
Kelley R. Hurst-Hess ◽  
Cheri A. Koetzner ◽  
Paul S. Masters
Keyword(s):  
Nucleocapsid Protein ◽  
Hepatitis Virus ◽  
Virus Assembly ◽  
Mouse Hepatitis Virus ◽  
M Protein ◽  
Growth Defect ◽  
S Protein ◽  
N Protein ◽  
Protein Incorporation ◽  
Carboxy Terminal

ABSTRACTThe coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.IMPORTANCEThe assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

Dehydroepiandrosterone, epiandrosterone and synthetic derivatives inhibit Junin virus replication in vitro

2008 ◽  
Vol 135 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Eliana G. Acosta ◽  
Andrea C. Bruttomesso ◽  
Juan A. Bisceglia ◽  
Mónica B. Wachsman ◽  
Lydia R. Galagovsky ◽  
...  
Keyword(s):  
Virus Replication ◽  
Junin Virus ◽  
Synthetic Derivatives ◽  
Junín Virus

scholarly journals Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic

2015 ◽  
Vol 90 (3) ◽  
pp. 1290-1297 ◽  
Author(s):  
Takaaki Koma ◽  
Michael Patterson ◽  
Cheng Huang ◽  
Alexey V. Seregin ◽  
Payal D. Maharaj ◽  
...  
Keyword(s):  
Mouse Model ◽  
Vaccine Strain ◽  
Causative Agent ◽  
Transmembrane Domain ◽  
Hemorrhagic Fever ◽  
Single Amino Acid ◽  
Junin Virus ◽  
Machupo Virus ◽  
Junín Virus

ABSTRACTMachupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that a MACV strain with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. Others and our group have identified the glycoprotein to be the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication and its ability to attenuate MACVin vivo. Recombinant MACV with the Candid#1 glycoprotein (rMACV/Cd#1-GPC) exhibited growth properties similar to those of Candid#1 and was genetically stablein vitro. In a mouse model of lethal infection, rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV strain expressing the glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model.IMPORTANCECurrently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrate for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.

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