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

Abstract

-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.Marek's disease virus / UL13 / pathogenesis / horizontal spread / chicken

Evolution and Diversity in Human Herpes Simplex Virus Genomes

“…This distinction earned it the alternative name of conserved herpesvirus-encoded protein kinase (CHPK). Loss of PK function attenuates viral growth in vitro and is deleterious for spread in vivo (121)(122)(123)(124)(125)(126)(127). The UL13 frameshift mutations in strains E06 and E25 are identical to each other and to one described previously in a subpopulation of HSV-1 strain F (29).…”

“…Strain E11 contains an alternate A 4 -to-A 3 mutation in UL13, which creates a frameshift within codon 319 and spares a portion of the kinase domain. Loss of PK due to a homopolymer-based frameshift in UL13 has also been observed in a bacterial artificial chromosome (BAC) clone of the fowl alphaherpesvirus Marek's disease virus (MDV) (125,126). Remarkably, the truncation of PK in MDV (at residue 176 of 513) occurs at a site similar to the truncation site in strains E06 and E25, again removing the kinase domain.…”

“…Culturing a virus necessarily removes a variety of selection pressures and bottlenecks present in vivo and introduces new selection pressures unique to cell growth in vitro. The HFMs and deletions described above are very likely to be deleterious for virus spread in humans (95)(96)(97)(98)(99)(121)(122)(123)(124)(125)(126)(127). This is likewise true for frameshifts and deletions recognized in previously sequenced strains (e.g., US9 in KOS or glycoprotein N [UL49.5] and UL56 in HF10) (47,135).…”

“…Remarkably, the truncation of PK in MDV (at residue 176 of 513) occurs at a site similar to the truncation site in strains E06 and E25, again removing the kinase domain. This truncation allows growth in culture but limits MDV transmission in vivo (125,126), suggesting that these PK truncations are selected for or amplified during passage in vitro.…”

Further Analysis of Marek's Disease Virus Horizontal Transmission Confirms That U L 44 (gC) and U L 13 Protein Kinase Activity Are Essential, while U S 2 Is Nonessential

“…The MDV U L 13 amino acid sequence contains the 11 (I to XI) conserved catalytic domains found in all protein kinases (11,37), and the protein is catalytically active (37). The transmission-deficient MDV previously analyzed contained a frameshift mutation within the U L 13 ORF leading to a truncated protein that encoded only the first two conserved domains (I and II) (2,17). We hypothesized that this truncated protein lacked kinase activity and that U L 13 kinase activity was important for transmission of MDV.…”