Differential processing of sindbis virus glycoprotein PE2 in cultured vertebrate and arthropod cells.
A step in the maturation of Sindbis virus glycoproteins is the cleavage of the precursor glycoprotein PE2 into E3 and E2 by furin or a furin-like host cell protease. The results presented here suggest that PE2 cleavage is an obligatory event for Sindbis virus maturation in C6/36 cells and demonstrate that certain mutants display a cell-specific PE2 cleavage phenotype. We previously have described Sindbis virus variants which fail to cleave PE2 because of incorporation of a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage site but are viable in BHK-21 cells by virtue of an additional mutation at E2 216 or E2 191 (TRSB-NE2G216 and TRSB-NE2T191, respectively) (H. W. Heidner, K. L. McKnight, N. L. Davis, and R. E. Johnston, J. Virol. 68:2683-2692, 1994). Other viable PE2 cleavage-defective mutants were constructed by substituting the parental residue at E2 position 1 (Arg), with Leu or Val (TRSB-E2L1 and TRSB-E2V1, respectively) (H.W. Heidner and R. E. Johnston, J. Virol. 68:8064-8070, 1994). When grown in BHK-21 cells, all four of these viruses replicated normally and incorporated PE2 in place of E2 in released virions. However, growth of TRSB-NE2G216 and TRSB-NE2T191 was severely restricted in cultured arthropod cells (C6/36 cells). Analysis of infected C6/36 cells by flow cytometry demonstrated that the restricted growth of TRSB-NE2G216 and TRSB-NE2T191 was not due to an impaired ability to initiate infection. In addition, TRSB-NE2G216 and TRSB-NE2T191 remained growth restricted in C6/36 cells following introduction of in vitro transcriptions by electroporation. In contrast, the PE2 cleavage defect of TRSB-E2L1 and TRSB-E2V1 was cell type specific. In C6/36 cells, the majority of PE2 was converted to E2, and these viruses replicated normally in C6/36 cells. These results demonstrated a consistent link between expression of a PE2 cleavage defect and restricted growth in C6/36 cells and suggest that cleavage of PE2 is required for maturation of Sindbis virus late in infection of C6/36 cells.
Heidner
H W
HW
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290, USA.
Knott
T A
TA
Johnston
R E
RE
D000330:Aedes / Q000166:cytology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012845:Sindbis Virus / Q000254:growth & development / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C022012:glycoprotein E1, Sindbis virus