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Journal of Virology, July 2006, p. 6771-6783, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Processing of Human Cytomegalovirus UL37 Mutant Glycoproteins in the Endoplasmic Reticulum Lumen prior to Mitochondrial Importation

Manohara S. Mavinakere ,1,{dagger},{ddagger} Chad D. Williamson,1,2,{dagger} Victor S. Goldmacher,3 and Anamaris M. Colberg-Poley1,2,4*

Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue NW, Washington, D.C. 20010,1 Department of Biochemistry and Molecular Biology, George Washington University, Washington, D.C. 20037,2 ImmunoGen, Inc., 128 Sidney Street, Cambridge, Massachusetts 02139,3 Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C. 200374

Received 9 March 2006/ Accepted 28 April 2006

The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues –1 and –3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic {alpha}-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37M, a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH2-terminal fragment, pUL37M-NH2, was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37M-COOH, was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH2-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37M, and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.

* Corresponding author. Mailing address: Children's Research Institute, Room 5720, Children's National Medical Center, 111 Michigan Ave. NW, Washington, DC 20010. Phone: (202) 884-3984. Fax: (202) 884-3929. E-mail: acolberg-poley{at}cnmcresearch.org.

{dagger} M.S.M. and C.D.W. contributed equally to this work.

{ddagger} Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.

Journal of Virology, July 2006, p. 6771-6783, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bozidis, P., Williamson, C. D., Colberg-Poley, A. M. (2008). Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells. J. Virol. 82: 2715-2726 [Abstract] [Full Text]  
  • Sharon-Friling, R., Goodhouse, J., Colberg-Poley, A. M., Shenk, T. (2006). Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores. Proc. Natl. Acad. Sci. USA 103: 19117-19122 [Abstract] [Full Text]  


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