Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

Whitt, Michael A.; Chong, L D; Rose, John K.

doi:10.1128/jvi.63.9.3569-3578.1989

Cited by 116 publications

(74 citation statements)

References 44 publications

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“…Plasmids encoding wild-type (pAR-G) (Whitt et al, 1989) and temperature-sensitive forms (pGtsO45-2/T7) (Gallione and Rose, 1985) of VSV G-protein were kindly provided by John Rose (Yale University, New Haven, CT) and Marino Zerial, respectively. Plasmids encoding rab5 (Chavrier et al, 1990), rab5N133I (Gorvel et al, 1991), rab7 (Chavrier et al, 1990), rab7N125I, rab9 (Lombardi et al, 1993), and rab9N124I were the kind gift of Marino Zerial.…”

Section: Infection/ Transf Ectionmentioning

confidence: 99%

“…Transient overexpression of recombinant VSV G protein was achieved by infecting BHK cells with a recombinant vaccinia virus expressing T7 RNA polymerase (Fuerst et al, 1986) and subsequent transfection with a plasmid encoding VSV G protein (Whitt et al, 1989) under the control of the T7 promoter (Fuerst et al, 1986). VSV G protein was readily detectable at the cell surface 5-6 h posttransfection and accessible to a monoclonal antibody added at 4°C (Fig.…”

Section: Vsv G Protein As An Endocytic Transport Markermentioning

confidence: 99%

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Rab 7: an important regulator of late endocytic membrane traffic.

Feng

Press

Wandinger‐Ness

1995

The Journal of Cell Biology

577

514

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Abstract. Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.

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Section: Infection/ Transf Ectionmentioning

confidence: 99%

Section: Vsv G Protein As An Endocytic Transport Markermentioning

confidence: 99%

Rab 7: an important regulator of late endocytic membrane traffic.

Feng

Press

Wandinger‐Ness

1995

The Journal of Cell Biology

577

514

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“…For expression in COS cells the mutated genes were cloned into the Xho I site of pJC119. For expression in HeLa cells the genes were cloned into a modified form of pAR2529 (Fuerst et al, 1986;Rosenberg et al, 1987) into which an Xho I site had been introduced at the Barn HI site (Whitt et al, 1989). Sequence changes in all mutated DNAs were confirmed by sequence analysis (Maxam and Gilbert, 1977;Sanger et al, 1977).…”

Section: Plasmid Constructions and Oligonucleotide-directed Mutagenesismentioning

confidence: 99%

Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum.

Zagouras

Rose

1989

The Journal of Cell Biology

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Abstract. The sequence Ser-Glu-Lys-Asp-Glu-Leu (SEKDEL) has been shown to be a signal which leads to retention of at least two proteins in the endoplasmic reticulum of animal cells (Munro and Pelham, 1987). In this study we tested the function of this signal by appending it to two secretory proteins, rat growth hormone and the ot subunit of human chorionic gonadotrophin (hCG-o0. We used oligonucleotidedirected mutagenesis and expression to generate proteins with SEKDEL added to the exact COOH termini and then carried out a detailed analysis of their transport in monkey COS cells. We found that transport was not blocked for either protein, but rather that the half-time for secretion was increased about sixfold for both proteins. Analysis of oligosaccharide processing on hCG-ot-SEKDEL and indirect immunofluorescence microscopy on cells expressing both proteins was consistent with a retardation of transport between the endoplasmic reticulum and the Golgi apparatus. A change in the last amino acid of the SEKDEL sequence from Leu to Val abolished the retardation almost completely, suggesting a highly specific interaction of the sequence with a receptor. A change in the first amino acid had little or no effect on retardation. We conclude that the SEKDEL signal can have strong effects on reducing the rate of protein exit from the endoplasmic reticulum without generating absolute retention. Presumably other features of protein structure must be important to generate absolute retention.M ECHANISMS exist for sorting of proteins within the exocytic pathway of eucaryotic cells. This sorting leads to retention of specific proteins within the ER and subcompartments of the Golgi apparatus, and is thought to be mediated by sorting signals in the proteins themselves and receptors in the pathway (reviewed by Rose and Doms, 1988). One of the simplest and clearest examples of a sorting signal is the tetrapeptide sequence Lys-Asp-Glu-Leu (KDEL) which Munro and Pelham (1987) noted at the COOH termini of four proteins that are residents of the ER. Deletion of this sequence from one of these resident ER proteins, GRP78, resuited in slow secretion of the truncated GRP78, and addition of the SEKDEL sequence from GRP78 to the secretory protein lysozyme resulted in retention of lysozyme in the ER. Although the receptor for the KDEL sequence has not yet been identified, it was suggested that it might reside in an early Golgi compartment and function through a recycling process that retrieves resident ER proteins back from the Golgi apparatus. Evidence for such a mechanism has been obtained both in mammalian cells (Pelham, 1988) and in yeast which use the related retention signal HDEL .Because the function of the KDEL signal had only been tested on one secretory protein from mammalian cells, we decided to test it further by appending it to two well-characterized secretory proteins that were under study in our laboratory, rat growth hormone (rGH), 1 and the ot subunit of human chorionic gonadotropin (hCG-o0. rGH is a nonglycosylated monomeric...

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“…Previous studies have noted that the length of cytoplasmic domain on VSV G, not the specific sequence, was required for efficient viral budding [19]. The real role of truncated G protein in attenuation of VSV has still not been identified, although it was reported that VSV C-terminal truncated G proteins acquired complex oligosaccharides ∼6-fold slower than for wt G protein and displayed a reduced rate of transport from the endoplasmic reticulum to the Golgi apparatus, and presumably to the cell surface [18].…”

Section: Inoculummentioning

confidence: 99%

“…The M protein is a multi-functional protein involved in virus assembly, budding and pathogenesis [15,16], and capable of inhibiting the transport of host mRNAs out of the nucleus significantly inhibiting type I interferon (IFN) signaling [17]. The G protein is responsible for viral binding to the host receptor and entry of VSV into host cells and its cytoplasmic domain is considered to play an important role in viral budding and packaging [18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of attenuated VSVs with mutated M or/and G proteins as vaccine vectors

Fang

Zhang

Sun

et al. 2012

Vaccine

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Vesicular stomatitis virus (VSV) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. In experimental animals (primates, rats, and mice), VSV has been shown to lead to neurotoxicities, such as hind limb paralysis. The virus matrix protein (M) and glycoprotein (G) are both major pathogenic determinants of wild-type VSV and have been the major targets for the production of attenuated strains. Existing strategies for attenuation included: (1) deletion or M51R substitution in the M protein (VSVΔM51 or VSVM51R, respectively); (2) truncation of the C-terminus of the G protein (GΔ28). Despite these mutations, recombinant VSV with mutated M protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant VSV with truncated G protein at high dose. Thus, a novel recombinant VSV (VSVΔM51-GΔ28) as well as other attenuated VSVs (VSVΔM51, VSV-GΔ28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. In vitro studies indicated that truncated G protein (GΔ28) could play a more important role than deletion of M51 (ΔM51) for attenuation of recombinant VSV. VSVΔM51-GΔ28 was determined to be the most attenuated virus with low pathogenicity in mice, with VSV-GΔ28 also showing relatively reduced pathogenicity. Further, neutralizing antibodies stimulated by VSV-GΔ28 proved to be significantly higher than in mice treated with VSVΔM51-GΔ28. In conclusion, among different attenuated VSVs with mutated M and/or G proteins, recombinant VSV with only truncated G protein (VSV-GΔ28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. These properties make VSV-GΔ28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease.

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Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

Cited by 116 publications

References 44 publications

Rab 7: an important regulator of late endocytic membrane traffic.

Rab 7: an important regulator of late endocytic membrane traffic.

Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum.

Evaluation of attenuated VSVs with mutated M or/and G proteins as vaccine vectors

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