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.
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)-alpha, 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.
Mandic
Lana
L
Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada.
Miller
Matthew S
MS
Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada.
Coulter
Corinne
C
Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada.
Munshaw
Brian
B
Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada.
Hertel
Laura
L
Department of Microbiology and Immunology, Health Sciences Addition HSA320, The University of Western Ontario, London, ON N6A 5C1, Canada.
D000595:Amino Acid Sequence; D017209:Apoptosis; D002460:Cell Line; D003587:Cytomegalovirus / Q000737:chemistry* / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005814:Genes, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D016601:RNA-Binding Proteins; C074546:US11 protein, herpesvirus; C118670:US2 protein, Varicellovirus; C456063:US9 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins