Analysis of the herpesvirus chemokine-binding glycoprotein G residues essential for chemokine binding and biological activity.

Viral chemokine-binding protein (vCKBP) are expressed by large DNA viruses, such as herpesviruses and poxviruses. vCKBP can bind chemokines with high affinity and efficiently neutralize their ability to induce cell migration. Recently, herpesvirus glycoprotein G (gG) was identified as a member of the vCKBP-4 subfamily. The structural domains of gG important for binding to chemokines and biological activity, however, are unknown. Here, we used equine herpesvirus type 1 (EHV-1) as a model to determine residues in EHV-1 gG that are involved in the processes of chemokine binding and interaction with target cells. First, comprehensive analysis of glycosylation of EHV-1 gG revealed that N-glycosylation is not required for binding of gG to chemokines but is essential for biological activity of the protein. Second, the epitope responsible for the binding to chemokines was localized to 40 amino acids in the hypervariable region (amino acids 301-340) of the protein. Third, hybrid molecules, designed as loss- and gain-of-function gG proteins, were engineered. In these hybrid glycoproteins the hypervariable regions of EHV-1 gG, a vCKBP, and the closely related EHV-4 gG, which does not display any chemokine binding capabilities, were exchanged. gG variants containing the EHV-1 hypervariable region were able to bind chemokines and were biologically active, whereas hybrid gGs containing the corresponding region of EHV-4 gG were not. Taking these results together, this report is the first to provide insight into the functional residues of an alphaherpesviral vCKBP.

A full UL13 open reading frame in Marek's disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that is highly contagious in poultry. Recombinant RB-1B (rRB-1B) reconstituted from an infectious genome cloned as a bacterial artificial chromosome (BAC) is unable to spread horizontally, quite in contrast to parental RB-1B. This finding suggests the presence of one or several mutations in cloned relative to parental viral DNA. Sequence analyses of the pRB-1B bacmid identified a one-nucleotide insertion in the UL13 orthologous gene that causes a frame-shift mutation and thereby results in a theoretical truncated UL13 protein (176 aa vs. 513 aa in parental RB-1B). UL13 genes are conserved among alphaherpesviruses and encode protein kinases. Using two-step "en passant" mutagenesis, we restored the UL13 ORF in pRB-1B. After transfection of UL13-positive pRB-1B DNA (pRB-1B*UL13), the resulting, repaired virus did not exhibit a difference in cell-to cell spread (measured by plaque sizes) and in UL13 transcripts in culture compared to parental rRB-1B virus. Although 89% of the chickens inoculated with rRB-1B*UL13 virus developed tumors in visceral organs, none of the contact birds did. MDV antigens were clearly expressed in the feather tips of rRB-1B infected chickens, suggesting that the UL13 gene mutation did not alter virus tropism of the feather follicle. The results indicate that the correction in UL13 gene alone is not sufficient to restore in vivo spreading capabilities of the rRB-1B virus, and that other region(s) of pRB-1B might be involved in the loss-of-function phenotype. This finding also shows for the first time that a full UL13 ORF is dispensable for MDV tumor formation and feather follicle tropism.