Functional Analysis of Potential Carboxy-Terminal Cleavage Sites of Tick-Borne Encephalitis Virus Capsid Protein

Abstract

The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Sitespecific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

Helices α2 and α3 of West Nile Virus Capsid Protein Are Dispensable for Assembly of Infectious Virions

“…A remarkable flexibility of protein C toward deletions and sequence alterations has already been observed in earlier studies (19,21,42,43,50). Protein C is essential for binding and packaging of genomic RNA and contributes to particle assembly and stability (reviewed in reference 38).…”

A trans -Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination

“…It is reasonable to assume that a single recombination event that gives rise to an infectious genome would produce at least 10 infectious particles and would thus outgrow the parental replicons within three passages or fewer. Flavivirus replicons typically multiply to 10 4 copies per cell (28,48), and because the experimental conditions used allowed multiple rounds of infection at each passage, we estimate that at least (Fig. 5), named recombinant 1 and recombinant 2, respectively.…”

Extension of Flavivirus Protein C Differentially Affects Early RNA Synthesis and Growth in Mammalian and Arthropod Host Cells

“…Ixodes ricinus embryo-derived cell line IRE/CTVM18 were cultured as described previously (5,35). For the production of TBEV from IRE/CTVM18 cells, the culture medium was supplemented with 20 mM HEPES, pH 7.4, and 0.6% 1 N NaOH.…”

“…To monitor RNA replication and RNA export following electroporation, BHK-21 cells were transfected with equimolar amounts of RNA (1.3 ϫ 10 12 RNA molecules), and the parameters were monitored by quantitative real-time PCR (qPCR) after reverse transcription of the viral RNA, as described previously (35).…”

“…Disturbance of these concerted cleavage events generally impairs (2,23,39) or even abrogates (19) virion production. Nevertheless, previous investigations of the C-terminal region of TBEV protein C revealed remarkable functional flexibility in the exact NS2B/3 cleavage position, as well as the cleavage substrate (35). In addition, several groups have also been able to supply protein C in trans to allow single-round infectious particles of several flaviviruses to be generated (10,13,27,36,40).…”