Aggregation and thermolability of some group V (G protein) and group III (M protein) mutants of vesicular stomatitis virus

Kellers, Petra; Uzgiris, E. E.; Cluxton, D.H.; Lenard, John

doi:10.1016/0042-6822(78)90158-7

Cited by 12 publications

(7 citation statements)

References 8 publications

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“…Although undetected by our conformational assays, at 39°C, subtle conformation change occurs in G protein independent of cellular factors since tsO45 viruses cannot infect cells at 39°C (Keller et al, 1978). Since the key event in virus entry Cells were labeled overnight with [35S]methionine, unincorporated radioisotope was removed, and the cells were infected with ts045 for 4 h. G protein was labeled with [35S]methionine for 10 min, and a fraction was shifted to 32°C for 30 min while the remainder was maintained for 30 min at 390C.…”

Section: Temperature-induced Changes In Mature G Triraersmentioning

confidence: 72%

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.

Silva

Balch

Helenius

1990

The Journal of Cell Biology

147

103

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Abstract. Parallel experiments in living cells and invitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39°C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32°C, these properties were reversed, and the protein was transported to the cell surface.Upon the shift up from 32°C back to 39°C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.

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Section: Temperature-induced Changes In Mature G Triraersmentioning

confidence: 72%

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.

Silva

Balch

Helenius

1990

The Journal of Cell Biology

147

103

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“…By contrast, infection at 320C by tsL512 (V) produces a number of lightdensity, noninfectious, glycoprotein-defective particles (data not shown). Stocks (propagated at 320C) of most known ts mutants in group V are inactivated by heating (9), and the four new ts (V) mutants are no exception (Table 8). Thus, the primary ts defect in these new mutants appear to be in the structure and/or maturation of the VSV glycoprotein, but further work is necessary to ascertain at what step biosynthesis is affected.…”

Section: Discussionmentioning

confidence: 99%

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein

Lodish¹,

Weiss²

1979

J Virol

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“…1) . It has previously been shown that similar heat treatment of infectious mutant virions results in loss of infectivity, a property called thermolability (11) . When the inhibitory capacity of several other thermolabile G protein mutants (ts 57, is 110, and tl 17) was tested, all showed good inhibition before heat treatment, whereas essentially no inhibitory activity remained after heat treatment (Table I) increased inhibition with preincubation continued for up to 2 h, with most of the increase occurring within 1 h. As shown in Fig.…”

Section: Differential Inhibition Of Infection By Heatinactivated Mutantsmentioning

confidence: 99%

Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step.

Miller¹,

Lenard²

1980

The Journal of Cell Biology

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In an assay measuring virus-directed RNA synthesis, infection of BHK cells by a standard test dose of vesicular stomatitis virus (VSV) was inhibited by ultraviolet light-irradiated wt VSV and by is 045, one of a number of thermolabile, temperature-sensitive G protein mutants of VSV . After heat treatment for 1 h at 45°C, the thermolabile mutants were no longer able to inhibit the VSV infection . In contrast, the thermolabile M protein mutant is G31 and the nonthermolabile G protein mutant is 044 could still inhibit the test VSV dose . Thus,the presence of G protein in its native conformation was necessary for inhibition of infection . There was little difference in the binding to cells or the internalization to a trypsinresistant state of is 045 or wt VSV before and after heat treatment, and there was no evidence of specific saturable receptors on the cell surface . None of the irradiated virions at concentrations that gave maximal inhibition of infection could prevent binding of infectious VSV to, or internalization by, BHK cells . The G protein-specific inhibition, therefore, did not occur at the cell surface but must have occurred at some intracellular site, which has been suggested to be the lysosome . The lysosomal inhibitor chloroquine, when added with the infecting virus, completely inhibited VSV infection at all multiplicities of infection tested, and it gave 50% inhibition when added 1 .5 h after infection . The possible importance of the lysosome in the intracellular pathway of infection is discussed. KEY WORDS vesicular stomatitis virus temperature-sensitive mutants -G protein thermolability virus binding -virus internalization chloroquineThe spike glycoprotein G of vesicular stomatitis virus (VSV)' has been shown to be essential to the ' Abbreviations used in this paper: VSV, vesicular stomatitis virus ; PBS, Dulbecco's phosphate-buffered saline ; MEM, Eagle's minimal essential medium; BES, N,Ninfectivity of VSV in BHK cells : both removal of the G protein by proteolysis (1) and coincubation of the cells with purified G protein in vesicular form (13) inhibit viral infection. It has been widely accepted, but not directly shown, that the importance of the G protein rests in its ability to enable VSV to attach to cells (2) . In this study, we have bis(2-hydroxyethyl)-2-aminoethane sulfonic acid; SFV, Semliki Forest virus ; BSA, bovine serum albumin; FCS, fetal calf serum ; m.o .i ., multiplicity of infection .J . CELL BIOLOGY

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Aggregation and thermolability of some group V (G protein) and group III (M protein) mutants of vesicular stomatitis virus

Cited by 12 publications

References 8 publications

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein

Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step.

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