Viral Serine/Threonine Protein Kinases

Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCEEHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines. E quine herpesvirus type 1 (EHV-1) and EHV-4 are members of the Herpesviridae family and Alphaherpesvirinae subfamily (1, 2). After initial replication in the upper respiratory tract, EHV-1 infects immune cells and migrates past the epithelial basement membrane to the lymph nodes and bloodstream (1-4). As a result, EHV-1 is able to spread throughout the body, where it infects endothelial cells (EC), causing vascular lesions an...

Section: Importancementioning

confidence: 99%

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a DynamicIn VitroModel

Spiesschaert

Goldenbogen

Taferner

et al. 2015

“…erpes simplex virus 1 (HSV-1) encodes at least two protein kinases, Us3 and UL13 (1)(2)(3)(4)(5)(6). HSV-1 Us3 is a serine/threonine protein kinase with an amino acid sequence that is conserved in the subfamily Alphaherpesvirinae (1,2).…”

mentioning

confidence: 99%

Phosphorylation of a Herpes Simplex Virus 1 dUTPase by a Viral Protein Kinase, Us3, Dictates Viral Pathogenicity in the Central Nervous System but Not at the Periphery

Shindo

Maruzuru

Kawaguchi

2014

Herpes simplex virus 1 (HSV-1) encodes Us3 protein kinase, which is critical for viral pathogenicity in both mouse peripheral sites (e.g., eyes and vaginas) and in the central nervous systems (CNS) of mice after intracranial and peripheral inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. We recently reported that Us3 phosphorylated HSV-1 dUTPase (vdUTPase) at serine 187 (Ser-187) in infected cells, and this phosphorylation promoted viral replication by regulating optimal enzymatic activity of vdUTPase. In the present study, we show that the replacement of vdUTPase Ser-187 by alanine (S187A) significantly reduced viral replication and virulence in the CNS of mice following intracranial inoculation and that the phosphomimetic substitution at vdUTPase Ser-187 in part restored the wild-type viral replication and virulence. Interestingly, the S187A mutation in vdUTPase had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. Similarly, the enzyme-dead mutation in vdUTPase significantly reduced viral replication and virulence in the CNS of mice after intracranial inoculation, whereas the mutation had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. These observations suggested that vdUTPase was one of the Us3 substrates responsible for Us3 pathogenicity in the CNS and that the CNS-specific virulence of HSV-1 involved strict regulation of vdUTPase activity by Us3 phosphorylation. IMPORTANCEHerpes simplex virus 1 (HSV-1) encodes a viral protein kinase Us3 which is critical for pathogenicity both in peripheral sites and in the central nervous systems (CNS) of mice following peripheral and intracranial inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. Here, we report that Us3 phosphorylation of viral dUTPase (vdUTPase) at serine 187 (Ser-187), which has been shown to promote the vdUTPase activity, appears to be critical for viral virulence in the CNS but not for pathogenic effects in peripheral sites. Since HSV proteins critical for viral virulence in the CNS are, in almost all cases, also involved in viral pathogenicity at peripheral sites, this phosphorylation event is a unique report of a specific mechanism involved in HSV-1 virulence in the CNS.

“…In addition, EBV PK may upregulate the expression of two viral proteins important for nuclear egress, BFRF1 and BFLF2 (11,33). Both EBV PK and the homologous HCMV kinase, UL97, greatly enhance but are not absolutely required for the release of infectious viral particles in vitro and appear to be intimately involved in the pathogenesis associated with viral infections in vivo (34,35). Although maribavir, an inhibitor of HCMV UL97, failed a phase III clinical trial in bone marrow transplant patients (36) (possibly due to insufficient dosing), CHPKs nevertheless remain very promising targets for development of novel antiviral therapeutics.…”

mentioning

confidence: 99%

Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in Epstein-Barr Virus-Infected Cells

Sun

Bristol

Iwahori

et al. 2013

H uman herpesviruses are enveloped viruses containing relatively large, double-stranded DNA genomes. Although all herpesviruses experience both latent and lytic stages of infection, they are grouped into three separate families (alpha-, beta-, and gammaherpesviruses) according to differences in sequence homology and cellular tropisms. The alphaherpesviruses, which comprise herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), cause recurrent skin lesions and meningitis (1, 2). Human cytomegalovirus (HCMV), human herpesviruses 6A and 6B (HHV6), and human herpesvirus 7 (HHV7) are betaherpesviruses, which cause severe disease in patients with compromised immune function (3, 4). The gammaherpesviruses are Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), which are causally associated with mononucleosis (EBV) as well as a variety of human cancers (5, 6).Each of the eight human herpesviruses encodes a protein kinase (PK) with discernible homology in amino acid sequences and positional similarity in their respective viral genomes. These related protein kinases, termed the conserved herpesvirus protein kinases (CHPKs), are important for viral replication and infection (7-13). They play important roles in multiple processes, including gene expression (8,11,14), viral DNA replication (11,(15)(16)(17), capsid nuclear egress (7,11,18,19), and the DNA damage response (20, 21). For example, EBV PK (the product of the BGLF4 gene) phosphorylates a number of different viral and cellular proteins, including the viral DNA polymerase processivity factor BMRF1 (7,(22)(23)(24); the latent viral proteins EBNA1 (25), EBNA2 (26), and EBNA LP (27); the EBV immediate early (IE) protein BZLF1 (28); the cell cycle regulatory proteins p27 (29) and pRB (30); nuclear lamin A/C (7, 31); and interferon regulatory factor 3 (IRF3) (32). In addition, EBV PK may upregulate the expression of two viral proteins important for nuclear egress, BFRF1 and BFLF2 (11,33). Both EBV PK and the homologous HCMV kinase, UL97, greatly enhance but are not absolutely required for the release of infectious viral particles in vitro and appear to be intimately involved in the pathogenesis associated with viral infections in vivo (34,35). Although maribavir, an inhibitor of HCMV UL97, failed a phase III clinical trial in bone marrow transplant patients (36) (possibly due to insufficient dosing), CHPKs nevertheless remain very promising targets for development of novel antiviral therapeutics.Two guanine nucleoside analogues, ganciclovir (GCV) and acyclovir (ACV), have been used frequently to inhibit replication of various human herpesviruses by targeting viral DNA polymerases (37-40). UL97 mediates the first step of GCV and ACV phosphorylation (41-43). Since the triphosphorylated forms of GCV and ACV are much better substrates for herpesvirus DNA polymerases than cellular DNA polymerases, GCV and ACV inhibit viral DNA replication more effectively than cellular DNA replication (44,45). It was recently found that EBV PK is req...