Polyprotein processing in caliciviruses

Bromfield, Annabel (2002) Polyprotein processing in caliciviruses. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Caliciviruses encode a single capsid protein, which in the Norwalk-like viruses (NLVs) and Vesiviruses are encoded in a separate reading frame (ORF) from the nonstructural proteins. Whereas in the Sapporo-like viruses (SLVs) and Lagoviruses they are encoded in a large ORF contiguous with the nonstructural proteins. Studies of the molecular biology and replication cycle of the human enteric caliciviruses has been severely hampered due to the inability to propagate these viruses in tissue culture. Stool samples from outbreaks are the primary source of viral RNA, however, the small quantities of stool available and the difficulties associated with manipulating RNA makes the construction of cDNA clones essential for in vitro studies and transfection of these viruses.

Mutations within a full length clone of Lordsdale NLV were corrected to produce an authentic full length clone and used in in vitro transcription and translation studies. These studies revealed a proteolytic pattern which suggested the presence of precursor proteins. These proteins were mapped using polyclonal antisera to defined regions of the polyprotein including the N terminal region. The N terminal region was expressed in an E. coli expression system and the recombinant proteins purified and used to raise hyperimmune antisera. In addition, an infectious full length clone of FCV(urbana) was adapted so that a guanosine nucleotide was engineered in front of the first 5' nucleotide to result in an increased efficiency of in vitro transcription and translation products in a rabbit reticulolysate system. Lordsdale NLV full length clone and Manchester SLV clones fused to GFP were used in transfection studies using a vaccinia T7 expression system. Transfection of these clones indicated that the nonstructural proteins are expressed in this system. Trans cleavage experiments were performed using full length clones of Manchester SLV, Southampton NLV, Lordsdale NLV, Jena virus and FCV against Manchester SLV, Southampton NLV and Jena NLV protease mutant clones. These studies showed that the NLVs are capable of trans cleavage, whereas the SLVs and FCV do not cleave in trans.