Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1.

Herpes simplex virus-1 (HSV-1), the prototype of the α-herpesvirus family, causes life-long infections in humans. Although generally associated with various mucocutaneous diseases, HSV-1 is also involved in lethal encephalitis. HSV-1 entry into host cells requires cellular receptors for both envelope glycoproteins B (gB) and D (gD). However, the gB receptors responsible for its broad host range in vitro and infection of critical targets in vivo remain unknown. Here we show that non-muscle myosin heavy chain IIA (NMHC-IIA), a subunit of non-muscle myosin IIA (NM-IIA), functions as an HSV-1 entry receptor by interacting with gB. A cell line that is relatively resistant to HSV-1 infection became highly susceptible to infection by this virus when NMHC-IIA was overexpressed. Antibody to NMHC-IIA blocked HSV-1 infection in naturally permissive target cells. Furthermore, knockdown of NMHC-IIA in the permissive cells inhibited HSV-1 infection as well as cell-cell fusion when gB, gD, gH and gL were coexpressed. Cell-surface expression of NMHC-IIA was markedly and rapidly induced during the initiation of HSV-1 entry. A specific inhibitor of myosin light chain kinase, which regulates NM-IIA by phosphorylation, reduced the redistribution of NMHC-IIA as well as HSV-1 infection in cell culture and in a murine model for herpes stromal keratitis. NMHC-IIA is ubiquitously expressed in various human tissues and cell types and, therefore, is implicated as a functional gB receptor that mediates broad HSV-1 infectivity both in vitro and in vivo. The identification of NMHC-IIA as an HSV-1 entry receptor and the involvement of NM-IIA regulation in HSV-1 infection provide an insight into HSV-1 entry and identify new targets for antiviral drug development.

Role of the herpes simplex virus 1 Us3 kinase phosphorylation site and endocytosis motifs in the intracellular transport and neurovirulence of envelope glycoprotein B.

Herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) in infected cells. This phosphorylation downregulates cell surface expression of gB and plays a role in viral pathogenesis in the mouse herpes stromal keratitis model. In the present study, we demonstrated that Us3 phosphorylation of gB Thr-887 upregulated the accumulation of endocytosed gB from the surfaces of infected cells. We also showed that two motifs in the cytoplasmic tail of gB, tyrosine at position 889 (Tyr-889) and dileucines at positions 871 and 872, were required for efficient downregulation of gB cell surface expression and upregulation of accumulation of endocytosed gB in infected cells. A systematic analysis of mutations in these three sequences in gB suggested that the expression of gB on the surfaces of infected cells was downregulated in part by the increase in the accumulation of endocytosed gB, which was coordinately and tightly regulated by the three gB trafficking signals. Tyr-889 appeared to be of predominant importance in regulating the intracellular transport of gB and was linked to HSV-1 neurovirulence in mice following intracerebral infection. These observations support the hypothesis that HSV-1 evolved the three gB sequences for proper regulation of gB intracellular transport and that this regulation plays a critical role in diverse aspects of HSV-1 pathogenesis.

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells.

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes.

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.