Langat virus M protein is structurally homologous to prM

Abstract

Langat (LGT) virus M protein has been generated in a recombinant system. Antiserum raised against the LGT virus M protein neutralizes tick-borne encephalitis serocomplex flaviviruses but not mosquito-borne flaviviruses, indicating that the M protein is exposed on the surface of virions. The antiserum recognizes intracellular LGT virus prM/M and binds to prM and M in Western blots of whole-cell lysates and purified virus, respectively. These data suggest that the prM and M proteins are structurally similar under native conditions and support the hypothesis that the "pr" portion of prM facilitates proper folding of the M protein for expression on the virion surface.

FIG. 1

M protein antiserum recognizes viral protein within the cytoplasm of LGT virus-infected Vero cells. (A) LGT virus-infected Vero cells fixed and probed at 3 days postinfection. (B) Mock-infected Vero cells. Cells probed with only the secondary antibody had no background fluorescence.

FIG. 2

Western blot analysis of LGT virus and virus-infected cell lysates. Wild-type LGT virus (TP21) was purified through a 20% sucrose gradient and run in 4 to 20% acrylamide gradient SDS-PAGE gels under both reducing (Red) and nonreducing (NR) conditions. Virus-infected cell lysates were run under denaturing nonreducing conditions in a 12% gel. Blots were probed with M protein antiserum. 9F9I and 9F9II are variant LGT viruses with mutations near the prM/E cleavage site that may alter the prM secondary structure (unpublished data).

FIG. 3

Sequence alignment of flavivirus M proteins. The viruses used for this analysis were LGT strain TP21 (GenBank protein sequence accession no. A41704), POW strain LB (Q04538), Louping Ill (LI) (NP044677), tick-borne encephalitis virus (TBE) strain HYPR (Q01299), DEN-4 (P09866), JE strain SA-14 (P27395), and YF 17D (GNWVY). Sequences were aligned using the AlignX alignment program in the VectorNTI sequence analysis package (InforMax).