Flavivirus West Nile (Sarafend) egress at the plasma membrane

Ng, Mah-Lee; Howe, J.; Sreenivasan, V.; Mulders, J.J.L.

doi:10.1007/bf01309477

Cited by 37 publications

(31 citation statements)

References 22 publications

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“…Our observations suggest that c-Yes may be involved in this process. In contrast, Ng et al observed formation of nucleocapsids at the plasma membrane as well as in large vesicles in Vero cells infected with WNV strain Sarafend (31,32). It is possible that these observations represent two distinct pathways by which the virus can exit the cell.…”

Section: Discussionmentioning

confidence: 58%

The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

Hirsch¹,

Medigeshi²,

Meyers³

et al. 2005

J Virol

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The role of cellular genes in West Nile virus (WNV) replication is not well understood. Examination of cellular transcripts upregulated during WNV infection revealed an increase in the expression of the src family kinase (SFK) c-Yes. WNV-infected cell lines treated with the SFK inhibitor PP2 demonstrated a 2-to 4-log decrease in viral titers, suggesting that SFK activity is required for completion of the viral replication cycle. RNA interference mediated knock-down of c-Yes, but not c-Src, and similarly reduced virus yield, specifically implicating c-Yes in WNV production. Interestingly, PP2 treatment did not reduce intracellular levels of either viral RNA or protein, suggesting that the drug does not act on the early stages of replication. However, endoglycosidase H (endoH) digestion of the viral envelope (E) glycoprotein revealed that the acquisition of endoH-resistant glycans by E, but not endogenous major histocompatibility complex class I, was reduced in PP2-treated cells, demonstrating that E specifically does not traffic beyond the endoplasmic reticulum in the absence of SFK activity. Electron microscopy further revealed that PP2-treated WNV-infected cells accumulated an increased number of virions in the ER compared to untreated cells. Therefore, we conclude that inhibition of SFK activity did not interfere with virus assembly but prevented transit of virions through the secretory pathway. These results identify c-Yes as a cellular protein that is involved in WNV assembly and egress.West Nile virus (WNV) is a newly emerging pathogen that has become a significant threat to the U.S. population. First detected in Uganda in 1937, the virus appeared in the United States in the summer of 1999, and since that time it has reemerged each year over an increasing geographical area (1). Typical of flaviviruses, WNV contains a single-stranded positive-sense 11-kb RNA genome. The genome is translated into a single polyprotein, which is cleaved by host and virus-encoded proteases into 10 functional subunits (5, 20). The WNV particle is composed of the subunits derived from the amino terminus of the polyprotein: capsid (C), precursor membrane/ membrane (prM/M), and envelope (E). The remaining seven proteins are nonstructural and function in virus replication. WNV infection results in proliferation and reorganization of intracellular membranes into several unique structures that colocalize with specific subsets of viral proteins, suggesting a distinct spatial segregation of the stages of virus replication (24, 48).WNV virion assembly appears to occur at the rough endoplasmic reticulum (ER), presumably by budding of the nucleocapsid (NC) into the ER lumen (5,20,28). In the context of viral infection, packaging of the genomic RNA requires NS2A and the replication of the RNA by the viral RNA-dependent RNA polymerase NS5 (16,21). The mechanism of NC budding into the ER and acquisition of the viral membrane and glycoproteins is unknown, although a hydrophobic region in the C protein has been proposed to be important in these ...

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Section: Discussionmentioning

confidence: 58%

The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

Hirsch¹,

Medigeshi²,

Meyers³

et al. 2005

J Virol

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“…These authors have shown that the characteristics of lipid-droplet-associated membranes in Huh-7 cells differ from those of ER membranes. In the case of flaviviruses, for which the mechanism of viral assembly and budding remains unclear (15), a few studies have demonstrated budding at the plasma membrane (13,36,37,41), and it has been proposed that the site of budding may be virus and cell type dependent (27). We demonstrate here that subpopulations of HCV structural proteins partition into cellular detergent-resistant, lipid-raft-like membrane fractions in HCVcc-producing cells (Fig.…”

Section: Discussionmentioning

confidence: 72%

Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

Aizaki¹,

Morikawa²,

Fukasawa

et al. 2008

J Virol

189

185

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In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.Hepatitis C virus (HCV) is recognized as a major cause of chronic liver disease, including chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. It presently affects approximately 200 million people worldwide (26). HCV is an enveloped positive-strand RNA virus belonging to the Hepacivirus genus of the family Flaviviridae. Its genome of ϳ9.6 kb encodes a polyprotein precursor of ϳ3,000 residues, and the structural proteins (core, E1, and E2) reside in its N-terminal region.Little is known about the assembly of HCV and its virion structure, because efficient production of authentic HCV particles has only recently been achieved. Nucleocapsid assembly generally involves oligomerization of the capsid protein and encapsidation of genomic RNA. This process is thought to occur upon interaction of the core protein with viral RNA, and this core-RNA interaction may induce a change from RNA replication to packaging. As with related viruses, the mature HCV virion likely consists of a nucleocapsid and an outer envelope composed of a lipid membrane and envelope proteins. Expression of the structural proteins in mammalian cells has been observed to generate virus-like particles with ultrastructural properties similar to those of HCV virions (5, 29). Packaging of these HCV-like particles into intracellular vesicles as a result of budding from the endoplasmic reticulum (ER) has also been observed (8,34). However, HCV structural proteins are observed both in the ER and in the Golgi apparatus (45). Moreover, complex N-linked glycans have been detected on the surfaces of HCV particles isolated from patient sera, suggesting that the glycans transit through the Golgi apparatus (44). Interactions between the core and E1/E2 proteins are thought to determine viral morphology and are mediated thro...

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“…Either cis or trans modes of maturation have been shown in several flaviviruses (e.g. WNV, DENV-2 and TBEV), depending on the particular virus strain and cells used (Leary & Blair, 1980;Hase et al, 1987;Ng, 1987;Ng & Hong, 1989;Ng et al, 1994aNg et al, , 2001Mackenzie et al, 1999; Šenigl et al, 2006). Šenigl et al (2006) described that TBEV exhibits a trans mode of maturation in mammalian cells, but a cis mode in tick cells.…”

Section: Discussionmentioning

confidence: 99%

Morphological changes in human neural cells following tick-borne encephalitis virus infection

Růžek

Vancová

Tesařová

et al. 2009

Journal of General Virology

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Tick-borne encephalitis (TBE) is one of the leading and most dangerous human viral neuroinfections in Europe and north-eastern Asia. The clinical manifestations include asymptomatic infections, fevers and debilitating encephalitis that might progress into chronic disease or fatal infection. To understand TBE pathology further in host nervous systems, three human neural cell lines, neuroblastoma, medulloblastoma and glioblastoma, were infected with TBE virus (TBEV). The susceptibility and virus-mediated cytopathic effect, including ultrastructural and apoptotic changes of the cells, were examined. All the neural cell lines tested were susceptible to TBEV infection. Interestingly, the neural cells produced about 100-to 10 000-fold higher virus titres than the conventional cell lines of extraneural origin, indicating the highly susceptible nature of neural cells to TBEV infection. The infection of medulloblastoma and glioblastoma cells was associated with a number of major morphological changes, including proliferation of membranes of the rough endoplasmic reticulum and extensive rearrangement of cytoskeletal structures. The TBEV-infected cells exhibited either necrotic or apoptotic morphological features. We observed ultrastructural apoptotic signs (condensation, margination and fragmentation of chromatin) and other alterations, such as vacuolation of the cytoplasm, dilatation of the endoplasmic reticulum cisternae and shrinkage of cells, accompanied by a high density of the cytoplasm. On the other hand, infected neuroblastoma cells did not exhibit proliferation of membranous structures. The virions were present in both the endoplasmic reticulum and the cytoplasm. Cells were dying preferentially by necrotic mechanisms rather than apoptosis. The neuropathological significance of these observations is discussed. INTRODUCTIONTick-borne encephalitis virus (TBEV) is the most medically important member of the tick-borne group of the genus Flavivirus within the family Flaviviridae (Thiel et al., 2005). The virus represents a causative agent of tick-borne encephalitis (TBE), a severe infection of the central nervous system (CNS) (Gritsun et al., 2003), which is prevalent throughout wide areas in Europe and Asia.The nature of the host cell response to TBEV infection remains undefined. Characteristic, but not disease-specific neuropathologic changes in the CNS include meningitis and polioencephalomyelitis predominantly in the spinal cord, brainstem and cerrebellum associated with inflammatory cell infiltration. These pathological changes have been described in mice (Osetowska & Wró blewskaMularczyk, 1966; Rů žek et al., 2009), hamsters (Simon et al., 1966), monkeys (Simon et al., 1967 and humans (Seitelberger & Jellinger, 1966; Környey, 1978;Beer et al., 1999; Schellinger et al., 2000; Gelpi et al., 2005 Gelpi et al., , 2006. Although cytopathic effects (CPEs) have been observed primarily in virus-infected neurons, other cells can also exhibit pathological changes, presumably through bystander injury.To clarify the inter...

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Flavivirus West Nile (Sarafend) egress at the plasma membrane

Cited by 37 publications

References 22 publications

The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

Morphological changes in human neural cells following tick-borne encephalitis virus infection

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