Abstract
ABSTRACTThe herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production.IMPORTANCE The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.
Highlights
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USAa; Department of Biological Sciences, College of Basic Sciences, Louisiana State University, Baton Rouge, Louisiana, USAb; Center for Computation & Technology, Louisiana State University, Baton Rouge, Louisiana, USAc
We have previously reported that pUL37 physically interacts with the membrane proteins pUL20 and glycoprotein K, the exact locations of the relevant gK and pUL37 binding sites remain unknown [26]. pUL20 and gK act as modulators of virus-induced fusion and interact with each other, in addition to binding the major Herpes simplex virus 1 (HSV-1) fusion protein gB, and pUL20 has recently been shown to interact with gM [30,31,32,33,34]
To delineate functional domains of the UL37 protein involved in infectious virus production, a cadre of UL37 gene mutations were generated by PCR-assisted mutagenesis targeting residues conserved among alphaherpesviruses
Summary
The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. These results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production.
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