Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease

Young Tae Ro, Scott M. Scheffter, Jean L. Patterson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid- polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.

Original languageEnglish (US)
Pages (from-to)8983-8990
Number of pages8
JournalJournal of Virology
Volume71
Issue number12
StatePublished - Dec 1997
Externally publishedYes

Fingerprint

RNA-directed RNA polymerase
RNA Replicase
Polyproteins
capsid
Cysteine Proteases
Capsid
Leishmania
Ribosomal Frameshifting
cysteine
proteinases
Viruses
antigens
Antigens
viruses
Parasites
pol Gene Products
parasites
antibodies
proteins
Antibodies

ASJC Scopus subject areas

  • Immunology

Cite this

Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease. / Ro, Young Tae; Scheffter, Scott M.; Patterson, Jean L.

In: Journal of Virology, Vol. 71, No. 12, 12.1997, p. 8983-8990.

Research output: Contribution to journalArticle

Ro, Young Tae ; Scheffter, Scott M. ; Patterson, Jean L. / Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease. In: Journal of Virology. 1997 ; Vol. 71, No. 12. pp. 8983-8990.
@article{a1536e8bcf494d75a9f69104284a85b4,
title = "Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease",
abstract = "Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid- polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.",
author = "Ro, {Young Tae} and Scheffter, {Scott M.} and Patterson, {Jean L.}",
year = "1997",
month = "12",
language = "English (US)",
volume = "71",
pages = "8983--8990",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease

AU - Ro, Young Tae

AU - Scheffter, Scott M.

AU - Patterson, Jean L.

PY - 1997/12

Y1 - 1997/12

N2 - Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid- polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.

AB - Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid- polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.

UR - http://www.scopus.com/inward/record.url?scp=0030730227&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030730227&partnerID=8YFLogxK

M3 - Article

C2 - 9371554

AN - SCOPUS:0030730227

VL - 71

SP - 8983

EP - 8990

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 12

ER -