Expression of the Conserved Herpesvirus Protein Kinase (CHPK) of Marek’s Disease Alphaherpesvirus in the Skin Reveals a Mechanistic Importance for CHPK during Interindividual Spread in Chickens

Abstract

The Herpesviridae encode many conserved genes, including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture to various degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek’s disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken to chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and Western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread and autophosphorylation of CHPK and that mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens, and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread. IMPORTANCE Marek’s disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek’s disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation but do not block interindividual spread from chicken to chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek’s disease while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined that MDV CHPK is not required for cell-to-cell spread, for disease induction, and for oncogenicity. However, it is required for interindividual spread, and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conservedHerpesviridaeprotein kinase (CHPK)

“…1C). Consistent with previous results, the relative level of CHPKmut was lower compared to CHPKwt under reducing conditions [23]. The relative amount of plaques produced from the virion preps by vCHPKmut was >35-fold lower compared to vCHPKwt samples (Fig.…”

“…Most CHPK-mutant or -null viruses tested are viable using traditional cell and tissue culture systems [6–10]. CHPKs have been linked to multiple processes during replication including nuclear egress [11–17], tegument association/dissociation [18, 19], viral gene expression [8, 12, 20–23], viral DNA replication [12, 16, 17, 24–29], DNA damage responses [30, 31], cell-cycle regulation [32–35], and evasion of the interferon response [36–38].…”

The Conserved Herpesviridae Protein Kinase (CHPK) of Gallid alphaherpesvirus 3 (GaHV3) Is Required for Horizontal Spread and Natural Infection in Chickens

“…An important question we were unable to address in this report is the effect of the CHPK mutation (K157A) on the expression of 301B/1 CHPK, since we do not have an antibody against 301B/1 CHPK. In the case of MDV CHPK, it has been shown that the overall protein level of CHPK is diminished, which has been attributed to protein stability [ 59 ]. This was shown using epitope tagged MDV CHPK, and the same approach could be applied in the future to address this question.…”

Coinfection in the host can result in functional complementation between live vaccines and virulent virus

“…DNA was extracted from infected FFE cells scraped from FFs or CEC cultures using the QIAamp DNA Mini Kit from Qiagen, LLC (Germantown, MD, USA) according to the manufacturer’s instructions. PCR was performed using DreamTaq Green 2× Master Mix (Thermo Fisher Scientific) and previously described primers to MDV gC and UL13 [ 23 , 24 ]. All PCR reactions were electrophoresed through 0.8% agarose gels and recorded using an Alpha Imager HP (Protein Simple) (Wallingford, CT, USA).…”