scholarly journals Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication

2007 ◽  
Vol 88 (10) ◽  
pp. 2800-2810 ◽  
Author(s):  
Michela Campagna ◽  
Mauricio Budini ◽  
Francesca Arnoldi ◽  
Ulrich Desselberger ◽  
Jorge E. Allende ◽  
...  
Keyword(s):  
Cytoplasmic Inclusion ◽  
Rna Replication ◽  
Viral Rna ◽  
Structural Protein ◽  
Cytoplasmic Inclusion Bodies ◽  
Replicative Cycle ◽  
In Cells

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1α is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1α is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1α was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1α is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.

scholarly journals Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

2008 ◽  
Vol 89 (7) ◽  
pp. 1770-1776 ◽  
Author(s):  
Chaozheng Zhang ◽  
Yueyong Liu ◽  
Liyue Liu ◽  
Zhiyong Lou ◽  
Hongyan Zhang ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Cytoplasmic Inclusion ◽  
Dwarf Virus ◽  
Structural Protein ◽  
Exact Function ◽  
Cytoplasmic Inclusion Bodies ◽  
Helical Protein ◽  
Spectroscopy Studies

Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, α-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.

scholarly journals Cytoplasmic Dynein Nucleates Microtubules to Organize Them into Radial Arrays In Vivo

2004 ◽  
Vol 15 (6) ◽  
pp. 2742-2749 ◽  
Author(s):  
Viacheslav Malikov ◽  
Anna Kashina ◽  
Vladimir Rodionov
Keyword(s):  
Cytoplasmic Dynein ◽  
Exact Function ◽  
Necessary And Sufficient ◽  
Stimulation Of ◽  
In Cells

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.

scholarly journals Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

2003 ◽  
Vol 77 (9) ◽  
pp. 5098-5108 ◽  
Author(s):  
Pablo Gastaminza ◽  
Beatriz Perales ◽  
Ana M. Falcón ◽  
Juan Ortín
Keyword(s):  
Influenza Virus ◽  
Rna Replication ◽  
Terminal Region ◽  
Viral Rna ◽  
Mrna Accumulation ◽  
Virus Rna ◽  
Transcription In Vitro ◽  
Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

scholarly journals In vitro and in vivo replication of influenza A H1N1 WSN33 viruses with different M1 proteins

2013 ◽  
Vol 94 (4) ◽  
pp. 884-895 ◽  
Author(s):  
Zhiguang Ran ◽  
Ying Chen ◽  
Huigang Shen ◽  
Xiaoxiao Xiang ◽  
Qinfang Liu ◽  
...  
Keyword(s):  
Influenza A ◽  
Cultured Cells ◽  
Important Determinant ◽  
Clinical Manifestations ◽  
Structural Protein ◽  
M1 Protein ◽  
Influenza A H1n1

The M1 protein is a major structural protein that has multiple functions in various steps within the life cycle of the influenza A virus (IAV). However, little is currently known about the role of M1 in IAV replication in vivo and the associated pathogenesis. In this study, six isogenic H1N1 WSN33 viruses, constructed to express unique M1 proteins derived from various strains, subtypes or WSN33 itself, were tested to determine in vitro and in vivo functional exchangeability of M1 proteins in the replication and pathogenesis of the WSN33 virus. Despite five chimeric M1 viruses replicating to levels similar to those of the parental WSN33 virus in cell cultures, all M1 chimeras exhibited improved replication and enhanced virulence in mice when compared with the WSN33 virus. Interestingly, M1 proteins derived from swine viruses caused more severe clinical diseases than those from human or quail. These data indicate that the M1 protein is an important determinant of viral replication and pathogenic properties in mice, although the functions of M1 observed in vivo are not adequately reflected in simple infections of cultured cells. Chimeric M1 viruses that are variable in their clinical manifestations described here will aid future understanding of the role of M1 in IAV pathogenesis.

scholarly journals Viability of Poliovirus/Rhinovirus VPg Chimeric Viruses and Identification of an Amino Acid Residue in the VPg Gene Critical for Viral RNA Replication

2003 ◽  
Vol 77 (13) ◽  
pp. 7434-7443 ◽  
Author(s):  
I. Wayne Cheney ◽  
Suhaila Naim ◽  
Jae Hoon Shim ◽  
Meghan Reinhardt ◽  
Bharati Pai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Rna Replication ◽  
Viral Rna ◽  
Sequencing Analysis ◽  
Virus Particles ◽  
Chimeric Rna ◽  
Chimeric Viruses

ABSTRACT Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3Dpol, in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3Dpol-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3Dpol was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3Dpol could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3Dpol to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu6 to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3Dpol in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.

scholarly journals Tyrosine 3 of Poliovirus Terminal Peptide VPg(3B) Has an Essential Function in RNA Replication in the Context of Its Precursor Protein, 3AB

2007 ◽  
Vol 81 (11) ◽  
pp. 5669-5684 ◽  
Author(s):  
Ying Liu ◽  
David Franco ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Rna Replication ◽  
Precursor Protein ◽  
Viral Rna ◽  
Hydroxyl Group ◽  
Wild Type ◽  
Replication Complex ◽  
A Genome ◽  
Essential Function

ABSTRACT Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3Dpol, but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3Dpol. Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.

MicroRNA-378-3p/5p suppresses the migration and invasiveness of oral squamous carcinoma cells by inhibiting KLK4 expression

2020 ◽  
Vol 98 (2) ◽  
pp. 154-163
Author(s):  
Zhi Cui ◽  
Shiqun Sun ◽  
Qilin Liu ◽  
Xuechun Zhou ◽  
Siyu Gao ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Squamous Carcinoma ◽  
Effective Measure ◽  
Mesenchymal Transition ◽  
Squamous Carcinoma Cells ◽  
Oral Squamous Carcinoma ◽  
In Cells

Distant metastasis frequently occurs in oral squamous cell carcinoma (OSCC) and contributes to the adverse prognosis for patients with OSCC. However, the potential mechanisms behind the metastasis have not yet been clarified. This study investigated the role of miR-378 in the migration and invasiveness of OSCC in vitro and in vivo. According to our results, the migration and invasiveness of OSCC cells were increased in cells overexpressing miR-378, and reduced in cells where miR-378-3p/5p was silenced. In addition, overexpression of miR-378 suppressed the expressions and activities of matrix metalloproteinase 9 (MMP-9) and MMP-2. Epithelial–mesenchymal transition (EMT) was restrained by overexpression of miR-378, as evidenced by an increase in E-cadherin expression and decrease in N-cadherin and uPA expression. However, knockdown of miR-378-3p/5p produced the opposite results. Moreover, kallikrein-related peptidase 4 (KLK4) was confirmed to be a target gene of miR-378. Overexpression of KLK4 reversed the induced decrease in migration and invasiveness of cells overexpressing miR-378 by upregulating the levels of MMP-9, MMP-2, and N-cadherin, and downregulating the level of E-cadhrin. Finally, the number of metastasis nodules in the lung tissues of nude mice was reduced by overexpression of miR-378, whereas the number of metastases increased with knockdown of miR-378. Taken together, our results suggest that the miR-378–KLK4 axis is involved in the mechanisms behind the migration and invasiveness of OSCC cells. Targeting the miR-378–KLK4 axis may be an effective measure for treating OSCC.

scholarly journals Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2162-2169 ◽  
Author(s):  
Saulius Serva ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Mass Spectrometry Analysis ◽  
Host Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Viral Replicase

ABSTRACT Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

scholarly journals SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication

2016 ◽  
Vol 90 (9) ◽  
pp. 4308-4319 ◽  
Author(s):  
Chan-I Su ◽  
Chung-Hsin Tseng ◽  
Chia-Yi Yu ◽  
Michael M. C. Lai
Keyword(s):  
Dengue Virus ◽  
Protein Stability ◽  
Posttranslational Modification ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Denv Infection ◽  
Cellular Protein ◽  
Viral Rna

ABSTRACTSmall ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular geneUbc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. Byin vivoandin vitroSUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication.IMPORTANCESUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.

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