Norwalk Virus N-Terminal Nonstructural Protein Is Associated with Disassembly of the Golgi Complex in Transfected Cells

Fernández-Vega, Virneliz; Sosnovtsev, Stanislav V.; Belliot, Gaël; King, Adriene D.; Mitra, Tanaji; Gorbalenya, Alexander E.; Green, Kim Y.

doi:10.1128/jvi.78.9.4827-4837.2004

Cited by 83 publications

(99 citation statements)

References 43 publications

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“…The membranous vesicles containing the calicivirus-like particles may have originated from a cellular organelle, as described for other members of the Caliciviridae family such as FCV (13). In in vitro experiments, the nonstructural N-terminal protein encoded by the 5Ј region of ORF1 for both GI (Norwalk virus) and GII (MD145 GII/4) NoVs was associated with Golgi apparatus localization, and there was a discrete time course in the disassembly of the Golgi complex into aggregates generating two patterns: one of marked discrete aggregates and the other a more diffuse distribution of the protein throughout the cytoplasm (10).…”

Section: Discussionmentioning

confidence: 99%

Pathogenesis of a Genogroup II Human Norovirus in Gnotobiotic Pigs

Cheetham

Souza

Meulia

et al. 2006

J Virol

257

264

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Human noroviruses (HuNoVs) are a major cause of foodborne gastroenteritis worldwide. Because they do not grow in cell culture and there is no animal model for HuNoVs, pathogenesis studies have been hampered. Thus, little is known about their replication strategies or induction of neutralizing antibodies.The limited information on their pathogenesis is from human volunteer studies of HuNoV infections in which villus atrophy in duodenal biopsies and presence of malabsorptive diarrhea were described (1,7,8). No information is available on lesions in other portions of the intestines of these volunteers (9). Intestinal transplant pediatric patients that were diagnosed with HuNoV infection developed secretory or osmotic diarrhea (19,20,31). These patients had prolonged diarrhea (17 to 326 days) due to immunosuppressive therapy. The detection of HuNoV RNA and the clinical symptoms remitted after reduction of the immunosuppressive therapy. Usually in exposed individuals, histologic lesions correlate with diarrhea, but in one report, lesions in volunteers who did not show clinical symptoms were described (42). There are also numerous reports of asymptomatic individuals who were infected with HuNoVs and shed virus in the feces (11, 28).Most past attempts to study these viruses in an animal model may have failed because (i) the human strains that were used were not closely related to the host animal NoV strains, (ii) sensitive detection techniques were lacking, and finally (iii) the role of histo-blood group antigen (HBGA) phenotypes in differential susceptibility of the host was unrecognized. Our goal was to adapt a HuNoV strain to replicate in the gnotobiotic (Gn) pig to develop an animal model for the study of HuNoV pathogenesis. Gnotobiotic pigs are good models for human enteric diseases (40) because pigs resemble humans in their gastrointestinal anatomy, physiology, and immune responses. The Gn pigs are immunocompetent at birth, but they lack maternal antibodies and previous or ongoing exposure to microbial agents, including caliciviruses.Recently, viral RNA genetically similar to that of human NoV GII (65 to 71% amino acid sequence identity in the capsid gene) was detected in pigs in Japan (46, 47) and Europe (22,48). In U.S. swine, our laboratory detected both viral RNA and virus particles similar to GII HuNoV (70% sequence identity in the capsid region) which were infectious for Gn pigs (50). Our approach to infect Gn pigs with a HuNoV was to use a GII strain that is closely related genetically to the identified GII porcine NoVs and that has a broad HBGA binding pattern because little information or reagents are available for pig HBGA. Additionally, we used sensitive assays and reagents including reverse transcription (RT)-PCR to detect fecal shedding, virus-like particles (VLPs) for serological assays, and antisera to these VLPs for antigen enzyme-linked immunosor-

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Section: Discussionmentioning

confidence: 99%

Pathogenesis of a Genogroup II Human Norovirus in Gnotobiotic Pigs

Cheetham

Souza

Meulia

et al. 2006

J Virol

257

264

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“…All except three insertions in this region were lost after the first two passages, suggesting that disruption of the putative transmembrane domain compromises viral fitness. The C-terminal end of the HuNoV NS1-2 equivalent protein (p48) ( Table 1) is also largely hydrophobic and contains a predicted TM domain between residues 361 and 379 (21,22). This TM domain is thought to potentially act as a membrane anchor and be involved in intracellular membrane rearrangements and Golgi apparatus disassembly (22).…”

Section: Resultsmentioning

confidence: 99%

“…The C-terminal end of the HuNoV NS1-2 equivalent protein (p48) ( Table 1) is also largely hydrophobic and contains a predicted TM domain between residues 361 and 379 (21,22). This TM domain is thought to potentially act as a membrane anchor and be involved in intracellular membrane rearrangements and Golgi apparatus disassembly (22). In contrast, the MNV NS1-2 localizes to the endoplasmic reticulum (ER) with no apparent Golgi association and promotes ER reorganization into distinct vesicular structures when transfected in isolation (30).…”

Section: Resultsmentioning

confidence: 99%

High-Resolution Functional Profiling of the Norovirus Genome

Thorne

Bailey

Goodfellow

2012

J Virol

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Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex. Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis in developed countries, with initial reports also indicating high incidence and mortality in developing countries (47). Due to the low infectious dose, multiple transmission routes, and high stability of HuNoV in the environment (41, 72), outbreaks typically occur in places with close living conditions, such as hospitals. Infections in hospitals alone cost the United Kingdom National Health Service an estimated £115 million and result in 47,000 lost bed days per year, compromising hospital services (39, 53). The majority of HuNoV infections are acute and self-resolving; however, links with conditions such as inflammatory bowel disease (36), infantile seizures (18), and neonatal necrotizing enterocolitis (65, 74) have been established. There are also increasing reports of persistent infections in the elderly and the immunocompromised, such as transplant recipients and some cancer patients (2,13,20,40,55,56).Details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. The discovery of murine norovirus (MNV) in 2003 has since facilitated studies on norovirus replication. Unlike HuNoV, MNV can be propagated in culture, displaying a tropism for macrophage and dendritic cells (75), and ca...

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“…Studies with Norwalk virus indicated that the p48 may interfere with disassembly of the Golgi complex and cellular protein trafficking (Ettayebi & Hardy, 2003;Fernandez-Vega et al, 2004) while studies with MNV and FCV point out that p48 is associated with the recruitment of ER membranes to the RC Hyde & Mackenzie, 2010). This recruitment of membranes is vital to the synthesis of new viral proteins by the actively replicating noroviruses (Hyde et al, 2009;Wobus et al, 2004).…”

Section: P48mentioning

confidence: 99%

“…The role of the remaining nonstructural proteins (NS1-2 and NS4) in norovirus replication is not yet well defined (Green, 2007). Available data suggests that NS1-2 and NS4, also known as N-term/p48 and p22, respectively, both contribute to norovirus replication complex formation on intracellular membranes, including that of the Golgi apparatus, and disrupt intracellular host protein trafficking (Ettayebi & Hardy, 2003;Fernandez-Vega et al, 2004;Hyde et al, 2009). …”

Section: Classification and Genome Organizationmentioning

confidence: 99%