Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production - PubMed (original) (raw)

Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production

E Lee et al. J Virol. 2000 Jan.

Abstract

Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.

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Figures

FIG. 1

Effect of the VPQAQA mutation in the signal sequence of prM on the efficiency of cleavage of prM in cell-free and transient expression assays. (A) COS-7 cells were transfected with eukaryotic expression plasmids encoding the YFV C, prM, and E proteins and with (pYF.VPQAQA) or without (pYF.s) the prM signal sequence mutation, infected with YFV (MOI, ∼10), or left untreated. At 2 days after transfection and 20 h after infection, the cells were metabolically labeled for 30 min and then chased for 1.5 h. Immunoprecipitation was done with anti-YFV hyperimmune ascitic fluid, and proteins were separated by SDS-PAGE (10% acrylamide). (B) Cell-free translation of YFV structural proteins was performed in the presence of microsomal membranes. Immunoprecipitation was done as described above, and proteins were analyzed by SDS-PAGE (12% acrylamide). Lanes 1 and 2 show translation products obtained with RNA transcribed in vitro from pYF.s and pYF.VPQAQA, respectively, and lane 3 shows a control transcription-translation-translocation reaction performed in the absence of plasmid DNA. (C) For VV vector expression of the YFV structural proteins with (VV.YF.VPQAQA) or without (VV.YF.s) the prM signal sequence mutation, CV1 cells were infected for 3 h (MOI, ∼10), starved for 30 min in methionine-free medium, and pulse-labeled for 15 min. The label was chased for the indicated times, and YFV proteins were immunoprecipitated and analyzed by SDS-PAGE (12% acrylamide). VV.TK− is a control virus which has no foreign DNA insert. Bands corresponding to YFV proteins are labeled on the left and sizes (in kilodaltons) of marker proteins are shown on right of the autoradiograms.

FIG. 2

YFV protein synthesis and viral replication in BHK cells transfected with in vitro-synthesized YFV 17D or VPQAQA RNA. BHK cells (107) were electroporated with approximately 1 μg of 17D or VPQAQA RNA and seeded in 60-mm petri dishes (2 × 106 cells/dish) for the kinetic study of virus-specific protein synthesis and virus replication. (A) Electroporated, YFV-infected (MOI, ∼1), or untreated cells were metabolically labeled for 3 h at various times after infection or transfection as shown. Immunoprecipitation was performed with YFV-specific hyperimmune ascitic fluid, and proteins were analyzed by SDS-PAGE (12% acrylamide). Sizes (in kilodaltons) of marker proteins are shown on the left, and viral proteins are labeled on the right. (B) Aliquots were taken at the indicated times from the culture supernatants of cells electroporated with 17D (triangles) or VPQAQA (squares) RNA, and virus titers were determined by plaque formation on Vero cells.

FIG. 3

Detection of YFV proteins in BHK cells transfected with 17D or VPQAQA RNA. BHK cells were electroporated with approximately 0.1 μg of 17D (A and B) or VPQAQA (C and D) RNA and seeded in a 24-well tray (4 × 104 cells/well). After incubation at 37°C for 6 h, culture medium from each well was replaced with fresh medium with (B and D) or without (A and C) ammonium chloride. Monolayers were fixed at 42 h posttransfection, and an immunofluorescence assay was performed with anti-YFV hyperimmune ascitic fluid. Areas in each well with the greatest density of fluorescent cells are shown.

FIG. 4

Effect of reversions and second-site mutations in the prM signal sequence on the efficiency of cleavage of prM in cell-free translation assays. Cell-free translation of YFV structural proteins was performed in the presence of microsomal membranes with appropriate RNA transcripts derived from plasmids pYF.s, pYF.VPQAQA, pYF.V2(P117→L), pYF.V4(H103→L), and pYF.V7(T107→I). Immunoprecipitation was performed with anti-YFV hyperimmune ascitic fluid, and proteins were separated by SDS-PAGE (12% acrylamide). Bands corresponding to YFV proteins E and prM are labeled on right.

FIG. 5

Virus titers of VPQAQA variants in vertebrate cells. (A) Vero cells (105) in 24-well dishes were infected (MOI, ∼0.1) with YFV 17D (squares) or VPQAQA variants V2(P117→L) (diamonds), V4(H103→L) (circles), and V7(T107→I) (triangles), and samples were taken between 24 and 72 h p.i. for assay of infectivity titers. (B) BHK cells were electroporated with full-length RNA transcripts (∼0.1 μg) incorporating the coding regions for the C-prM junctions of V2(P117→L), V4(H103→L), V7(T107→I), and YFV 17D. Electroporated cells (2 × 106) were plated in 60-mm dishes, and supernatant samples were taken between 16 and 64 h posttransfection for assay of infectivity titers. Data for 17D RNA (squares), V2′(P117→L) RNA (diamonds), V4′(H103→L) RNA (circles), and V7′(T107→I) RNA (triangles) are shown.

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Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production - PubMed (original) (raw)