scholarly journals Phosphorylation of Rubella Virus Capsid Regulates Its RNA Binding Activity and Virus Replication

2003 ◽  
Vol 77 (3) ◽  
pp. 1764-1771 ◽  
Author(s):  
Lok Man J. Law ◽  
Jason C. Everitt ◽  
Martin D. Beatch ◽  
Charles F. B. Holmes ◽  
Tom C. Hobman
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Binding ◽  
Virus Assembly ◽  
Binding Activity ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Positive Strand Rna

ABSTRACT Rubella virus is an enveloped positive-strand RNA virus of the family Togaviridae. Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.

scholarly journals Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis

2000 ◽  
Vol 74 (11) ◽  
pp. 5133-5141 ◽  
Author(s):  
Yuying Liang ◽  
Shirley Gillam
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Synthesis ◽  
Rnase Protection Assay ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

scholarly journals Strand-Specific RNA Synthesis Defects in a Poliovirus with a Mutation in Protein 3A

2003 ◽  
Vol 77 (23) ◽  
pp. 12679-12691 ◽  
Author(s):  
Natalya L. Teterina ◽  
Mario S. Rinaudo ◽  
Ellie Ehrenfeld
Keyword(s):  
Rna Synthesis ◽  
Binding Activity ◽  
Viral Rna ◽  
Wild Type ◽  
Positive Strand ◽  
Polyprotein Processing ◽  
Delayed Onset ◽  
Methionine Residue ◽  
Cell Extracts ◽  
Positive Strand Rna

ABSTRACT Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.

scholarly journals VP40 Octamers Are Essential for Ebola Virus Replication

2005 ◽  
Vol 79 (3) ◽  
pp. 1898-1905 ◽  
Author(s):  
Thomas Hoenen ◽  
Viktor Volchkov ◽  
Larissa Kolesnikova ◽  
Eva Mittler ◽  
Joanna Timmins ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Rna Binding ◽  
Virus Assembly ◽  
Wild Type Virus ◽  
Wild Type ◽  
Hek 293 ◽  
Significant Difference

ABSTRACT Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.

scholarly journals A Recombinant Influenza A Virus Expressing anRNA-Binding-Defective NS1 Protein Induces High Levels of BetaInterferon and Is Attenuated inMice

2003 ◽  
Vol 77 (24) ◽  
pp. 13257-13266 ◽  
Author(s):  
Nicola R. Donelan ◽  
Christopher F. Basler ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Mdck Cells ◽  
Binding Activity ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Dsrna Binding

ABSTRACT Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-β) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-β production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able to replicate in MDCK cells to titers close to those of wild-type virus. This mutant virus had intermediate virulence in mice, between those of the wild-type and parental NS1 R38AK41A viruses. These results suggest not only that the IFN antagonist properties of the NS1 protein depend on its ability to bind dsRNA but also that they can be modulated by amino acid residues not involved in RNA binding.

scholarly journals The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines

2018 ◽  
Vol 92 (21) ◽  
Author(s):  
Keisuke Nakagawa ◽  
Krishna Narayanan ◽  
Masami Wada ◽  
Vsevolod L. Popov ◽  
Maria Cajimat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Virus Assembly ◽  
Host Gene ◽  
Wild Type Virus ◽  
Rna Cleavage ◽  
Host Gene Expression ◽  
Biological Functions ◽  
Wild Type ◽  
Infected Cells

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induceIFN-β andIFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.IMPORTANCEMERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

scholarly journals Production of Noncapped Genomic RNAs is Critical to Sindbis Virus Disease and Pathogenicity

2020 ◽  
Author(s):  
Autumn T. LaPointe ◽  
V. Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Viral Replication ◽  
Sindbis Virus ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Genomic Rnas ◽  
The Brain

ABSTRACTAlphaviruses are positive-sense RNA viruses that utilize a 5’ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to wild type infection. The mice infected with the D355A nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both capping mutants had roughly equivalent viral titer in the brain compared to wild type virus, illustrating that the changes in mortality were not due to deficits in viral replication or dissemination. Examination of the brain tissue revealed that mice infected with the D355A capping mutant had significantly reduced cell death and immune cell infiltration compared to the N376A mutant and wild type virus. Finally, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aid in viral pathogenesis.AUTHOR SUMMARYMosquito transmitted alphaviruses have been the cause of widespread outbreaks of disease which can range from mild illness to lethal encephalitis or severe polyarthritis. In order to successfully replicate, the alphavirus RNA genome needs a 5’ cap structure so that the genome can be translated and produce the viral replication machinery. Despite this, a large number of viral genomes produced during infection do not have a 5’ cap structure, and their role during infection is unknown. Using mouse models of infection and point mutations in the nsP1 protein of Sindbis virus which alter the amount of noncapped genomic RNA (ncgRNA) produced, we found the decreasing the production of ncgRNA greatly reduced morbidity and mortality as well as proinflammatory cytokine expression, resulting in less tissue-damaging inflammation in the brain. These studies suggest that the ncgRNAs contribute to pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events

2006 ◽  
Vol 80 (14) ◽  
pp. 6917-6925 ◽  
Author(s):  
LokMan J. Law ◽  
Carolina S. Ilkow ◽  
Wen-Pin Tzeng ◽  
Matthew Rawluk ◽  
David T. Stuart ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Rubella Virus ◽  
Rna Binding ◽  
Virus Assembly ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Direct Role ◽  
Virus Capsid ◽  
Early Replication ◽  
Virus Capsid Protein

ABSTRACT The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.

scholarly journals Cell-Dependent Role for the Poliovirus 3′ Noncoding Region in Positive-Strand RNA Synthesis

2004 ◽  
Vol 78 (3) ◽  
pp. 1344-1351 ◽  
Author(s):  
David M. Brown ◽  
Steven E. Kauder ◽  
Christopher T. Cornell ◽  
Gwendolyn M. Jang ◽  
Vincent R. Racaniello ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Mutant Virus ◽  
Noncoding Region ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Virus Growth ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.

scholarly journals Effects of Defined Mutations in the 5′ Nontranslated Region of Rubella Virus Genomic RNA on Virus Viability and Macromolecule Synthesis

1998 ◽  
Vol 72 (1) ◽  
pp. 641-650 ◽  
Author(s):  
Konstantin V. Pugachev ◽  
Teryl K. Frey
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Point Mutations ◽  
Wild Type Virus ◽  
Multiple Point ◽  
Genomic Rna ◽  
Reading Frame ◽  
Viral Phenotype ◽  
Nontranslated Region ◽  
Different Strains

ABSTRACT The 5′ end of the genomic RNA of rubella virus (RUB) contains a 14-nucleotide (nt) single-stranded leader (ss-leader) followed by a stem-and-loop structure [5′(+)SL] (nt 15 to 65), the complement of which at the 3′ end of the minus-strand RNA [3′(−)SL] has been proposed to function as a promoter for synthesis of genomic plus strands. A second intriguing feature of the 5′ end of the RUB genomic RNA is the presence of a short (17 codons) open reading frame (ORF) located between nt 3 and 54; the ORF encoding the viral nonstructural proteins (NSPs) initiates at nt 41 in an alternate translational frame. To address the functional significance of these features, we compared the 5′-terminal sequences of six different strains of RUB, with the result that the short ORF is preserved (although the coding sequence is not conserved) as is the stem part of both the 5′(+)SL and 3′(−)SL, while the upper loop part of both structures varies. Next, using Robo302, an infectious cDNA clone of RUB, we introduced 31 different mutations into the 5′-terminal noncoding region, and their effects on virus replication and macromolecular synthesis were examined. This mutagenesis revealed that the short ORF is not essential for virus replication. The AA dinucleotide at nt 2 and 3 is of critical importance since point mutations and deletions that altered or removed both of these nucleotides were lethal. None of the other mutations within either the ss-leader or the 5′(+)SL [and accordingly within the 3′(−)SL], including deletions of up to 15 nt from the 5′(+)SL and three different multiple-point mutations that lead to destabilization of the 5′(+)SL, were lethal. Some of the mutations within both ss-leader and the 5′(+)SL resulted in viruses that grew to lower titers than the wild-type virus and formed opaque and/or small plaques; in general mutations within the stem had a more profound effect on viral phenotype than did mutations in either the ss-leader or upper loop. Mutations in the 5′(+)SL, but not in the ss-leader, resulted in a significant reduction in NSP synthesis, indicating that this structure is important for efficient translation of the NSP ORF. In contrast, viral plus-strand RNA synthesis was unaffected by the 5′(+)SL mutations as well as the ss-leader mutations, which argues against the proposed function of the 3′(−)SL as a promoter for initiation of the genomic plus-strand RNA.

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