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Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis

Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada

Correspondence
Laura Hertel
lhertel{at}uwo.ca

The human cytomegalovirus (CMV) US2–US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2–US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection.

A supplementary table, listing construction details of the expression vectors generated in this study using PCR, is available with the online version of this paper.