Functional analysis of virion host shutoff protein of pseudorabies virus.
During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.
Lin
Hui-Wen
HW
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Kuo-Kuang Road, Taichung 402, Taiwan.
Chang
Yuan-Yen
YY
Wong
Min-Liang
ML
Lin
Jen-Wei
JW
Chang
Tien-Jye
TJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D011558:Herpesvirus 1, Suid / Q000201:enzymology / Q000502:physiology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012260:Ribonucleases