Expression of the La Crosse M Segment Proteins in a Recombinant Vaccinia Expression System Mediates pH-Dependent Cellular Fusion

Jacoby, David; Cooke, Charles L.; Prabakaran, Indira; Boland, Jocelyn; Nathanson, Neal; González‐Scarano, Francisco

doi:10.1006/viro.1993.1213

Cited by 30 publications

(27 citation statements)

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“…Mutations in CT domains affect cell surface expression of BUNV glycoproteins. Detection of viral glycoproteins on the surfaces of infected cells has been reported for some bunyaviruses, such as hantaviruses (35,41), LACV (20), and Uukuniemi virus (26). Here the role of the CT domains of both the Gn and Gc proteins in cell surface expression of BUNV glycoproteins was investigated by either immunofluorescence or a biotin-labeling technique.…”

Section: Resultsmentioning

confidence: 99%

“…For many viruses, including bunyaviruses, the mildly acidic milieu within the late endosomes is required for the fusion process (7). In addition, many viruses, including several bunyaviruses, have been reported to mediate syncytium formation (fusion from within) under low-pH conditions (2,17,20,29). Computational analyses predicted the Gc glycoproteins of bunyaviruses to be low-pH-dependent class II fusion proteins (15).…”

Section: Discussionmentioning

confidence: 99%

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Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Shi

Kohl

et al. 2007

J Virol

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The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Shi

Kohl

et al. 2007

J Virol

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“…Huh7 cells grown on coverslips were cotransfected with 1 g pI.18-LACVM and 0.5 g pEGFP-C1 (Clontech) using 4 l DAC-30 transfection reagent (Eurogentec) in 200 l serum-free medium (OptiMEM; Gibco-BRL). At 16 h posttransfection, the DMEM was replaced with fusion buffer (Earle's balanced salt solution without sodium bicarbonate, 10 mM HEPES, 0.2% bovine serum albumin, pH 5.5) for 5 min (19). Cells were washed three times with phosphate-buffered saline and incubated in DMEM with 10% fetal calf serum for another 4 h at 37°C.…”

Section: Cells and Virusesmentioning

confidence: 99%

Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

Blakqori

Weber

2005

J Virol

103

105

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La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3 ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10 8 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSsexpressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

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“…Gc is therefore involved in the hemagglutination inhibition (12,14,17) and also in viral entry, playing a critical role in the fusion between the viral envelope and the cellular membrane (23,25). Gc undergoes a pH-dependent conformational change associated with cell-to-cell and virus-to-cell fusion (10,11,15,22). Gn protein is poorly immunogenic (12,18), but it is required for targeting Gc to Golgi apparatus from the endoplasmic reticulum (5).…”

mentioning

confidence: 99%

“…Gn protein is poorly immunogenic (12,18), but it is required for targeting Gc to Golgi apparatus from the endoplasmic reticulum (5). Furthermore, a role in cell-to-cell fusion cannot be ruled out (15,24) and Gn is also thought to be the protein responsible for viral attachment in the mosquito midgut (20). Gc contains virus-specific epitopes that could be used in the typing of California serogroup viruses, whereas N protein seems to be more conserved (12,13).…”

mentioning

confidence: 99%

Prevalence and Protein Specificity of Human Antibodies to Inkoo Virus Infection

Putkuri

Vaheri

Vapalahti

2007

Clin Vaccine Immunol

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Inkoo virus (INKV),In contrast, in patients with long-standing immunity, the Gc response was more prominent and the N response was weaker. In conclusion, a diagnostic IgG IFA pattern distinguishing between acute infection and longstanding immunity was observed. N protein seems to be the optimal antigen for the serodiagnosis of acute infection, and the Gc protein could be appropriate for the serosurveillance of INKV.

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Expression of the La Crosse M Segment Proteins in a Recombinant Vaccinia Expression System Mediates pH-Dependent Cellular Fusion

Cited by 30 publications

References 0 publications

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

Prevalence and Protein Specificity of Human Antibodies to Inkoo Virus Infection

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