scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

2016 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notable feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double stranded RNA intermediate in the cytoplasm of the infected cell which is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5’ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterisation of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

2016 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notable feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double stranded RNA intermediate in the cytoplasm of the infected cell which is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5’ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterisation of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2134 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of theCaliciviridaefamily of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater

2007 ◽  
Vol 74 (2) ◽  
pp. 477-484 ◽  
Author(s):  
Jinhee Bae ◽  
Kellogg J. Schwab
Keyword(s):  
Cell Culture ◽  
Surface Water ◽  
Multiple Linear Regression Model ◽  
Viral Persistence ◽  
Environmental Stability ◽  
Great Promise ◽  
Feline Calicivirus ◽  
Virus Infectivity ◽  
Murine Norovirus ◽  
Surface Water And Groundwater

ABSTRACT Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log10/day for FCV, 0.13 and 0.10 log10/day for PV, 0.12 and 0.06 log10/day for MS2, and 0.09 and 0.05 log10/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log10/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log10/day) and MNV (0.04 ± 0.03 log10/day) and were significantly different from those of FCV (0.08 ± 0.03 log10/day) and PV (0.09 ± 0.03 log10/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.

Surrogates for the Study of Norovirus Stability and Inactivation in the Environment: A Comparison of Murine Norovirus and Feline Calicivirus

2006 ◽  
Vol 69 (11) ◽  
pp. 2761-2765 ◽  
Author(s):  
JENNIFER L. CANNON ◽  
EFSTATHIA PAPAFRAGKOU ◽  
GEUNWOO W. PARK ◽  
JASON OSBORNE ◽  
LEE-ANN JAYKUS ◽  
...  
Keyword(s):  
Cell Culture ◽  
Thermal Inactivation ◽  
Genetic Relatedness ◽  
Mammalian Cell Culture ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Log Reduction ◽  
Culture Model ◽  
Dry Conditions ◽  
Ph Range

Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values <3 and >9. FCV was more stable than MNV-1 at 56°C, but both viruses exhibited similar inactivation at 63 and 72°C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4°C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.

scholarly journals Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle

2006 ◽  
Vol 80 (4) ◽  
pp. 1653-1661 ◽  
Author(s):  
Clara C. Posthuma ◽  
Danny D. Nedialkova ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Jeroen H. Blokhuis ◽  
Alexander E. Gorbalenya ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Pleiotropic Effects ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Genome Replication ◽  
Conserved Residues

ABSTRACT The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.

Hydrogen Peroxide Vapor Decontamination in a Patient Room Using Feline Calicivirus and Murine Norovirus as Surrogate Markers for Human Norovirus

2016 ◽  
Vol 37 (5) ◽  
pp. 561-566 ◽  
Author(s):  
Torsten Holmdahl ◽  
Mats Walder ◽  
Nathalie Uzcátegui ◽  
Inga Odenholt ◽  
Peter Lanbeck ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Tissue Culture ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Patient Room ◽  
Human Norovirus ◽  
Log Reduction ◽  
Infective Dose ◽  
Hydrogen Peroxide Vapor

OBJECTIVETo determine whether hydrogen peroxide vapor (HPV) could be used to decontaminate caliciviruses from surfaces in a patient room.DESIGNFeline calicivirus (FCV) and murine norovirus (MNV) were used as surrogate viability markers to mimic the noncultivable human norovirus. Cell culture supernatants of FCV and MNV were dried in triplicate 35-mm wells of 6-well plastic plates. These plates were placed in various positions in a nonoccupied patient room that was subsequently exposed to HPV. Control plates were positioned in a similar room but were never exposed to HPV.METHODSVirucidal activity was measured in cell culture by reduction in 50% tissue culture infective dose titer for FCV and by both 50% tissue culture infective dose titer and plaque reduction for MNV.RESULTSNeither viable FCV nor viable MNV could be detected in the test room after HPV treatment. At least 3.65 log reduction for FCV and at least 3.67 log reduction for MNV were found by 50% tissue culture infective dose. With plaque assay, measurable reduction for MNV was at least 2.85 log units.CONCLUSIONSThe successful inactivation of both surrogate viruses indicates that HPV could be a useful tool for surface decontamination of a patient room contaminated by norovirus. Hence nosocomial spread to subsequent patients can be avoided.Infect Control Hosp Epidemiol 2016;37:561–566

scholarly journals Mutagenesis of Tyrosine 24 in the VPg Protein Is Lethal for Feline Calicivirus

2004 ◽  
Vol 78 (9) ◽  
pp. 4931-4935 ◽  
Author(s):  
Tanaji Mitra ◽  
Stanislav V. Sosnovtsev ◽  
Kim Y. Green
Keyword(s):  
Amino Acids ◽  
Cdna Clone ◽  
Mutational Analysis ◽  
Covalent Bond ◽  
Feline Calicivirus ◽  
Tyrosine Residues ◽  
Positive Sense ◽  
Growth Properties ◽  
Calculated Mass ◽  
Covalently Linked

ABSTRACT The genome of feline calicivirus (FCV) is an ∼7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3′ end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5′ end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

Recovery and Disinfection of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, from Hard Nonporous and Soft Porous Surfaces

2015 ◽  
Vol 78 (10) ◽  
pp. 1842-1850 ◽  
Author(s):  
THOMAS YEARGIN ◽  
ANGELA FRASER ◽  
GUOHUI HUANG ◽  
XIUPING JIANG
Keyword(s):  
Sodium Hypochlorite ◽  
Limit Of Detection ◽  
Plaque Assay ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Surface Type ◽  
Log Reduction ◽  
Porous Surfaces

Human norovirus is a leading cause of foodborne disease and can be transmitted through many routes, including environmental exposure to fomites. In this study, both the recovery and inactivation of two human norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), on hard nonporous surfaces (glass) and soft porous surfaces (polyester and cotton) were evaluated by both plaque assay and reverse transcription quantitative PCR method. Two disinfectants, sodium hypochlorite (8.25%) and accelerated hydrogen peroxide (AHP, at 4.25%) were evaluated for disinfection efficacy. Five coupons per surface type were used to evaluate the recovery of FCV and MNV by sonication and stomaching and the disinfection of each surface type by using 5 ml of disinfectant for a contact time of 5 min. FCV at an initial titer of ca. 7 log PFU/ml was recovered from glass, cotton, and polyester at 6.2, 5.4, and 3.8 log PFU/ml, respectively, compared with 5.5, 5.2, and 4.1 log PFU/ml, respectively, for MNV with an initial titer of ca. 6 log PFU/ml. The use of sodium hypochlorite (5,000 ppm) was able to inactivate both FCV and MNV (3.1 to 5.5 log PFU/ml) below the limit of detection on all three surface types. AHP (2,656 ppm) inactivated FCV (3.1 to 5.5 log PFU/ml) below the limit of detection for all three surface types but achieved minimal inactivation of MNV (0.17 to 1.37 log PFU/ml). Reduction of viral RNA by sodium hypochlorite corresponded to 2.72 to 4.06 log reduction for FCV and 2.07 to 3.04 log reduction for MNV on all three surface types. Reduction of viral RNA by AHP corresponded to 1.89 to 3.4 log reduction for FCV and 0.54 to 0.85 log reduction for MNV. Our results clearly indicate that both virus and surface types significantly influence recovery efficiency and disinfection efficacy. Based on the performance of our proposed testing method, an improvement in virus recovery will be needed to effectively validate virus disinfection of soft porous surfaces.

scholarly journals Development of a reverse-genetics system for murine norovirus 3: long-term persistence occurs in the caecum and colon

2012 ◽  
Vol 93 (7) ◽  
pp. 1432-1441 ◽  
Author(s):  
Armando Arias ◽  
Dalan Bailey ◽  
Yasmin Chaudhry ◽  
Ian Goodfellow
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Reverse Genetics ◽  
Viral Rna ◽  
Viral Gastroenteritis ◽  
Murine Norovirus ◽  
Wild Type ◽  
Adaptive Mutations ◽  
Viral Sequences

Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide, yet, due to the inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo because it establishes persistence in wild-type mice, yet causes symptoms resembling gastroenteritis in immune-compromised STAT1−/− mice. The lack of reverse-genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least 56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence were detected in the caecum and colon, suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host, providing new insights into the biology of norovirus infections.

Akt plays differential roles during the life cycles of acute and persistent murine norovirus strains in macrophages

2021 ◽  
Author(s):  
Irene A. Owusu ◽  
Karla D. Passalacqua ◽  
Carmen Mirabelli ◽  
Jia Lu ◽  
Vivienne Young ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Strain ◽  
Virus Assembly ◽  
Viral Assembly ◽  
Akt Signaling ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Cellular Processes ◽  
Infectious Cycle ◽  
Virus Strains

Akt (Protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes such as glucose metabolism and cell proliferation for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or diminished cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. Importance Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity; yet there are no small animal models supporting productive HNoV infection, or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

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