Herpesvirus Pathogenesis – Tegument Proteins

Investigator:

• Sylvia van den Hurk

Background:

Bovine herpesvirus-1 (BHV-1) causes a number of diseases worldwide in cattle and is an important factor in shipping fever. Infectious bovine rhinotracheitis caused by BHV-1 can cause death in up to 10 per cent of infected animals. In addition, BHV-1 contributes to abortion, stillbirths and calf mortality.  Existing vaccines may have adverse effects on immune responses overall or are not capable of generating strong, long-lasting immunity to the virus. BHV-1 is classified in the subfamily of alphaherpesviruses and has similarities to herpes simplex virus and varicella zoster virus in humans.

A unique feature of the herpes virion is the presence of a complex compartment, the tegument, located between the nucleocapsid and the virus envelope. After virus entry the tegument proteins are released into the cytoplasm of infected cells, which allows them to play an essential role in establishing conditions for efficient viral replication. Later in infection, the tegument proteins participate in the morphogenesis of mature progeny virions, although the molecular mechanisms underlying the formation of the tegument region are largely undefined for BHV-1. Various regulatory functions, including modulation of transcription, kinase activity, RNase activity, DNA packaging and immune evasion have been attributed to tegument proteins.

To gain a better understanding of BHV-1 replication, which may ultimately enable the development of more effective and safer vaccines, we intend to characterize the functions of and interactions between the major tegument proteins of BHV-1, in particular VP8, VP22, US3 and U13.

Objectives:

1. Determine whether tegument proteins, in particular VP8, VP22, US3 and UL13, are essential for the viral infectious process in vitro and in vivo, and if so, which stage of the viral replication cycle is affected.
2. Examine the role of tegument proteins, VP8, VP22, US3 and UL13, in virion morphogenesis.
3. Assess the role of tegument proteins in innate and adaptive responses to BHV-1 in vitro and in vivo. 

Progress:

VP8 is the most abundant component of mature virions in BHV-1 and a target of the humoral and cellular immune response. We showed that VP8 exhibits a steady-state nuclear localization at early stages of infection and during transient expression suggesting a functional role in the nucleus. VP8 possesses nuclear localization and export signals, enabling it to shuttle between the nucleus and cytoplasm.  Although VP8 is clearly phosphorylated, the responsible kinases had not been identified. We observed interactions between VP8 and two kinases, CK2 and US3, and showed that VP8 is phosphorylated by both of these proteins. A further characterization of US3 demonstrated the presence of two critical residues: lysine 195 within the ATP binding pocket and lysine 282 within the catalytic loop. Interestingly, a BHV-1 U_L47 deletion mutant exhibited more than 100-fold decreased viral titers in vitro, and the U_L47 gene product was indispensable for BHV-1 replication in vivo. These results suggest that some unknown function of VP8 may be critical for BHV-1 viability.

VP22 associates with the nuclear matrix, binds chromatin and is capable of intercellular spread. We confirmed that VP22 has intercellular trafficking properties in live cells and mapped the nuclear localization and shuttling functions of VP22, by identifying a functional NLS and NES. VP22-YFP expressed during BHV-1 infection translocated to the nucleus through three different pathways: early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage. The VP22 ¹³¹PRPR¹³⁴ NLS was not required for the late massive nuclear translocation, but essential for the targeting of VP22 to discrete dot-like nuclear domains. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection, which may be critical for its diverse functions.

Future Direction:

BHV-1 can suppress the immune system at various levels. For instance, BHV-1 induces apoptosis of CD4+ Th cells, which impairs cytokine production and the development of both humoral and cell-mediated immune responses. The ability of BHV-1 to down-regulate MHC class I expression compromises the development of CTL responses against BHV-1 as well as other pathogens involved in bovine respiratory disease.  Several proteins play a role in the modulation of the innate and adaptive responses to BHV-1. The gene products of bICP0, U_L41, U_L49.5 and U_S4 can diminish the innate or adaptive response to a number of herpesviruses including BHV-1, while UL13 and US3 down-regulate the IFN response of other herpesviruses, at least ex vivo.

We will generate deletions to obtain further insight into the roles of these proteins in the innate and adaptive response to BHV-1 infection in vivo. Such deletion mutants of BHV-1 have applications as vaccine candidates. Furthermore, therapeutics that target tegument proteins and interrupt initiation of infection or virus assembly, in particular through inhibiting phosphorylation-dependent nuclear transport of viral gene products, may be developed.