Sharon K Fodor | Search PubMed

PubMed2002

Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive.

Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus that replicates naturally in fish at temperatures near 4 degrees C. The reverse transcriptase (RT) protein from virus particles isolated from walleye tumours was purified and biochemically characterized. Like the RT of the distantly related murine leukaemia virus, WDSV RT sediments as a monomer in the absence of template. It exhibits a K(m) of 22 microM for TTP in an assay with poly(rA) as a template and oligo(dT) as a primer. The enzyme is rapidly inactivated at temperatures greater than 15 degrees C. The ratio of RT activity at 15 degrees C to that at 4 degrees C is similar for WDSV and recombinant human immunodeficiency virus type 1, suggesting that, at least with this template, the fish enzyme is not specially adapted to function more efficiently in the cold.

Animals↗

PubMed2002

Characterization of the protease of a fish retrovirus, walleye dermal sarcoma virus.

Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4 degrees C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from Escherichia coli as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.

Amino Acid Sequence↗