Conserved herpesvirus protein kinases

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task

A Live-Attenuated HSV-2 ICP0− Virus Elicits 10 to 100 Times Greater Protection against Genital Herpes than a Glycoprotein D Subunit Vaccine

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry's lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0− virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0− virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein

Phosphorylation of the Epstein-Barr Virus (EBV) DNA Polymerase Processivity Factor EA-D by the EBV-Encoded Protein Kinase and Effects of the l-Riboside Benzimidazole 1263W94

A member of the family of l-riboside benzimidazole compounds, 1263W94, was shown recently to inhibit replication of Epstein-Barr virus (EBV) (V. L. Zacny, E. Gershburg, M. G. Davis, K. K. Biron, and J. S. Pagano, J. Virol. 73:7271–7277, 1999). In the present report the effect of 1263W94 on the phosphorylation pattern of the EBV DNA polymerase processivity factor, EA-D, during viral reactivation in latently EBV-infected Akata cells is analyzed. This pattern specifically changes with progression of cytolytic infection. In the presence of 1263W94 the appearance of the hyperphosphorylated form of EA-D is mainly affected. Next, coexpression of the cloned EBV-encoded protein kinase (EBV PK), BGLF4, with EA-D demonstrated the ability of EBV PK to phosphorylate EA-D to its hyperphosphorylated form in transient assays. However, the phosphorylation of EA-D was not directly inhibited by 1263W94 in these coexpression assays. The results indicate that the EBV PK appears to be responsible for the hyperphosphorylation of EA-D, imply that the phosphorylation status of EA-D is important for viral replication, and suggest that 1263W94 acts at a level other than direct inhibition of EA-D phosphorylation by EBV PK

Hyperphosphorylation of EBNA2 by Epstein-Barr Virus Protein Kinase Suppresses Transactivation of the LMP1 Promoter

The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle

Effects of Maribavir and Selected Indolocarbazoles on Epstein-Barr Virus Protein Kinase BGLF4 and on Viral Lytic Replication

The human cytomegalovirus (HCMV) homolog of the Epstein-Barr virus (EBV) protein kinase (PK), UL97, is inhibited by maribavir (1263W94) and selected indolocarbazoles. Here we show that only one of these indolocarbazoles (K252a), but not maribavir, inhibits autophosphorylation of the EBV PK, BGLF4. However, maribavir and another indolocarbazole, NGIC-I, do inhibit EBV DNA synthesis, suggesting that although these last compounds inhibit both HCMV and EBV, they seem to operate through differ-ent pathways

Pan-HSV-2 IgG antibody in vaccinated mice and guinea pigs correlates with protection against herpes simplex virus 2.

We lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2's 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 (-) viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes

The Durability of Vaccine Efficacy against Ocular HSV-1 Infection Using ICP0 Mutants 0∆NLS and 0∆RING Is Lost over Time

Vaccines to viral pathogens in experimental animal models are often deemed successful if immunization enhances resistance of the host to virus challenge as measured by cumulative survival, reduction in virus replication and spread and/or lessen or eliminate overt tissue pathology. Furthermore, the duration of the protective response against challenge is another important consideration that drives a vaccination regimen. In the current study, we assessed the durability of two related vaccines, 0∆NLS and 0∆RING, against ocular herpes simplex virus type 1 (HSV-1) challenge in mice thirty days (short-term) and one year (long-term) following the vaccine boost. The short-term vaccine efficacy study found the 0∆RING vaccine to be nearly equivalent to the 0∆NLS vaccine in comparison to vehicle-vaccinated mice in terms of controlling virus replication and preserving the visual axis. By comparison, the long-term assessment of the two vaccines found notable differences and less efficacy overall as noted below. Specifically, the results show that in comparison to vehicle-vaccinated mice, the 0∆NLS and 0∆RING vaccinated groups were more resistant in terms of survival and virus shedding following ocular challenge. Moreover, 0∆NLS vaccinated mice also possessed significantly less infectious virus in the peripheral and central nervous systems but not the cornea compared to mice vaccinated with vehicle or 0∆RING which had similar levels. However, all vaccinated groups showed similar levels of blood and lymphatic vessel genesis into the central cornea 30 days post infection. Likewise, corneal opacity was also similar among all groups of vaccinated mice following infection. Functionally, the blink response and visual acuity were 25–50% lower in vaccinated mice 30 days post infection compared to measurements taken prior to infection. The results demonstrate a dichotomy between resistance to infection and functional performance of the visual axis that collectively show an overall loss in vaccine efficacy long-term in comparison to short-term studies in a conventional prime-boost protocol

The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions

<div><p>Herpes simplex virus type 1 (HSV-1) encodes two <i>bona fide</i> serine/threonine protein kinases, the <i>US3</i> and <i>UL13</i> gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of <i>UL13</i> and <i>US3</i> genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.</p></div