Role of the UL45 protein in herpes simplex virus entry via low pH-dependent endocytosis and its relationship to the conformation and function of glycoprotein B

Abstract

Herpesviruses commandeer distinct cellular pathways to enter target cells. The mechanism by which herpes simplex virus (HSV) selects a pH-dependent, endocytic route or a pH-independent route remains to be elucidated. We investigated the role of the non-glycosylated viral envelope protein UL45 in HSV entry via endocytosis. UL45 plays a role in mediating cell-cell fusion and has been proposed to functionally interact with gB to regulate membrane fusion. Thus, we also probed the impact of UL45 on the structure and function of gB present in virions. A UL45 deletion virus successfully entered cells via low pH, endocytic pathway with wild type kinetics. In the absence or presence of UL45, the antigenic conformation of virion gB appeared unaltered. Antibodies to gB neutralized infection of the UL45-deletion virus and wild type virus to a similar extent, regardless of whether the target cells supported low pH endocytic or non-endocytic entry routes. Lastly, HSV virions were inactivated by low pH regardless of the presence of UL45. The results, together with previous studies, suggest that UL45 plays distinct roles in cell-cell fusion and virus-cell fusion during acid-dependent entry. Keywordsherpes simplex virus; viral endocytosis; UL45; glycoprotein B Herpes simplex virions can use pH-dependent endocytosis to initiate infection of host cells (Nicola et al., 2003). Other human herpesviruses have also been shown to utilize low pH, endocytic pathways for entry (Akula et al., 2003;Finnen et al., 2006;Ryckman et al., 2006). HSV may use an acid-dependent pathway to enter mucosal epithelial cells at the portal of entry in the human host . Infection of neurons isthought to occur via a pHindependent mechanism (Lycke et al., 1988;Nicola et al., 2005;Stiles et al., 2008). Direct, acid-independent penetration at the cell surface is the entry pathway in Vero cells (Wittels and Spear, 1991), which are a commonly used cell type to study HSV biology. The utilization of multiple cellular pathways to enter different physiologically relevant cell types is an emerging theme among herpesviruses (Frampton et al., 2007;Miller and Hutt-Fletcher, 1992 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Wang and Shenk, 2005), distinct viral envelope protein complexes determine target cell tropism and entry pathway. For HSV, glycoproteins gB, gD, and gH-gL are required for entry via pH-independent (Spear, 1993) and pH-dependent (Nicola and Straus, 2004) pathways. We theorize that one or more specific HSV envelope proteins direct the virus to the low pH endosomal route. Several HSV envelope proteins ar...

Contributions of Herpes Simplex Virus 1 Envelope Proteins to Entry by Endocytosis

“…2B). Together with previous results (23), this suggests that envelope proteins gE, gG, gI, gJ, gM, UL45p, and Us9p are individually dispensable for entry into two cell lines that support endocytic entry. The possibility that one or more nonessential proteins affect the rate of entry by endocytosis pathways cannot be ruled out.…”

“…4). Together with previous results (23), this suggests that gE, gG, gI, gJ, gM, UL45p, and Us9p do not determine the sensitivity of HSV to acid inactivation. The target of low-pH inactivation may be one or more components of the required entry machinery, gB, gD, and gH-gL.…”

“…Envelope proteins that are required for replication in Vero cells have been deemed "essential," and those that are dispensable are termed "nonessential" (19)(20)(21)(22). With the exception of gC and UL45p (17,23), the contribution of the nonessential envelope proteins to the endocytic entry process has not been evaluated.…”

Evolution and Diversity in Human Herpes Simplex Virus Genomes

“…Four proteins unique to alphaherpesviruses are also highly conserved (UL20, UL45, VP13-14 [UL47], and gK [UL53]). The functions of two of these, UL20 and UL45, are not well known, and their conservation indicates that further investigation of their function is warranted (138)(139)(140)(141)(142). A third protein, VP13-14 (UL47) modulates transactivator VP16 (UL48) to induce high levels of immediate early gene expression (143,144).…”

“…A third protein, VP13-14 (UL47) modulates transactivator VP16 (UL48) to induce high levels of immediate early gene expression (143,144). The proteins UL20, UL45, and gK have been proposed to interact with each other and with the herpesvirus fusion machinery (138)(139)(140)(141)(142).…”