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.

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 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.

scholarly journals Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov

2015 ◽  
Vol 46 (3) ◽  
pp. 861-865 ◽  
Author(s):  
Sivam Visnuvinayagam ◽  
Thangavel K ◽  
Lalitha N ◽  
Malmarugan S ◽  
Kuppannan Sukumar
Keyword(s):  
Cell Culture ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Virus Strain ◽  
Newcastle Disease ◽  
Newcastle Disease Virus Strain

scholarly journals Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells

2019 ◽  
Vol 4 ◽  
pp. 82 ◽  
Author(s):  
Harriet V. Mears ◽  
Edward Emmott ◽  
Yasmin Chaudhry ◽  
Myra Hosmillo ◽  
Ian G. Goodfellow ◽  
...  
Keyword(s):  
Viral Protein ◽  
Innate Immune ◽  
Interferon Response ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Murine Macrophage ◽  
Viral Protein Synthesis ◽  
Viral Translation ◽  
Double Stranded Rna

Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro. Results: Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.

Serum-free cell culture system supplemented with lipid-rich albumin for hepatitis C virus (strain O of genotype 1b) replication

2007 ◽  
Vol 125 (2) ◽  
pp. 162-168 ◽  
Author(s):  
Ken-ichi Abe ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Hiromichi Dansako ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Strain ◽  
Culture System ◽  
Free Cell ◽  
Cell Culture System ◽  
Serum Free ◽  
Genotype 1B

scholarly journals Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

2020 ◽  
Vol 48 (1) ◽  
Author(s):  
Hiroka Aonuma ◽  
Itoe Iizuka-Shiota ◽  
Tokio Hoshina ◽  
Shigeru Tajima ◽  
Fumihiro Kato ◽  
...  
Keyword(s):  
Virus Strain ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Virus Disease ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Virus Strains

Abstract Background Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. Results Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present. Conclusions The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

Mutational Events in Consecutive Passages of Hepatitis A Virus Strain GBM during Cell Culture Adaptation

Virology ◽  
1994 ◽  
Vol 204 (1) ◽  
pp. 60-68 ◽  
Author(s):  
Judith Graff ◽  
Christa Kasang ◽  
Andrea Normann ◽  
Mechtild Pfisterer-Hunt ◽  
Stephen M. Feinstone ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Strain ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Culture Adaptation

scholarly journals ER-Shaping Atlastin Proteins Act as Central Hubs to Promote Flavivirus Replication and Virion Assembly

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 31
Author(s):  
Christopher J Neufeldt ◽  
Mirko Cortese ◽  
Pietro Scaturro ◽  
Berati Cerikan ◽  
Jeremy Wideman ◽  
...  
Keyword(s):  
Viral Genome ◽  
Viral Assembly ◽  
Host Factors ◽  
Genome Replication ◽  
Membrane Structures ◽  
Virion Assembly ◽  
Virus Particles ◽  
Membrane Invagination ◽  
Viral Genome Replication ◽  
Flavivirus Infection

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are formed through membrane invagination into the endoplasmic reticulum (ER) lumen, termed invaginated vesicles or vesicle packets (VPs), as well as large areas of bundled smooth ER, termed convoluted membranes. Though the morphology of these virus-induced membrane structures has been well characterized, the viral and host constituents that make up flaviviral ROs are still poorly understood. Here, we identified a subset of ER resident proteins (atlastins), normally required for maintaining ER tubule networks, as critical host factors for flavivirus infection. Specific changes in atlastin (ATL) levels had dichotomous effects on flaviviruses with ATL2 depletion, leading to replication organelle defects and ATL3 depletion to changes in viral assembly/release pathways. These different depletion phenotypes allowed us to exploit virus infection to characterize non-conserved functional domains between the three atlastin paralogues. Additionally, we established the ATL interactome and show how it is reprogrammed upon viral infection. Screening of specific ATL interactors confirmed non-redundant ATL functions and identified a role for ATL3 in vesicle trafficking. Our data demonstrate that ATLs are central host factors that coordinate the ER network and shape the ER during flavivirus infection.

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