Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces the rapid onset of T-cell lymphomas in poultry. The MDV-encoded oncoprotein Meq plays an important role in oncogenicity, as its deletion abolishes the ability of the virus to induce tumours. It has been shown previously that Meq oncogenicity is linked to its interaction with C-terminal binding protein 1 (CtBP), a property also shared by other virus-encoded oncoproteins such as adenovirus E1A and Epstein–Barr virus EBNA3A and -3C. Therefore, this study examined whether Meq also shares the properties of these viral oncoproteins in interacting with other binding partners such as heat-shock protein 70 (Hsp70), a molecular chaperone protein linked to multiple cellular functions including neoplastic transformation. Confocal microscopic analysis demonstrated that MDV infection induced nuclear accumulation of Hsp70 and its co-localization with Meq. Biochemical evidence of Meq–Hsp70 interaction was obtained by two-way immunoprecipitation with Meq- and Hsp70-specific antibodies. To demonstrate further the Meq–Hsp70 interaction in virus-induced lymphomas, recombinant MDV was generated expressing an N-terminal tandem affinity purification (TAP) tag-fused Meq by mutagenesis of the infectious BAC clone of the oncogenic MDV strain RB-1B. Demonstration of Hsp70 in the TAP-tag affinity purified Meq from tumours induced by the recombinant virus, using quadrupole time-of-flight tandem mass spectrometry analysis, further confirmed the Meq–Hsp70 interaction in the transformed lymphocytes. Given the well-documented evidence of the tumorigenic properties of Hsp70 and its interaction with a number of other known viral oncoproteins, demonstration of the interaction of Meq and Hsp70 is significant in MDV oncogenesis.
The complete DNA sequence of the Marek's disease virus serotype 1 vaccine strain CVI988 was determined and consists of 178 311 bp with an overall gene organization identical to that of the oncogenic strains. In examining open reading frames (ORFs), nine differ between vaccine and oncogenic strains. A 177 bp insertion was identified in the overlapping genes encoding the Meq, RLORF6 and 23 kDa proteins of CVI988. Three ORFs are predicted to encode truncated proteins. One, designated 49.1, overlaps the gene encoding the large tegument protein UL36 and encodes a severely truncated protein of 34 aa. The others, ORF5.5/ORF75.91 and ORF3.0/78.0, located in the repeat regions (diploid), encode a previously unidentified ORF of 52 aa and a truncated version of the virus-encoded chemokine (vIL-8), respectively. Subtle genetic changes were identified in the two ORFs encoding tegument proteins UL36 and UL49. Only one diploid ORF (ORF6.2/ORF75.6) present in the genomes of the three virulent strains is absent in the CVI988-BAC genome. Seventy non-synonymous amino acid substitutions were identified that could differentiate CVI988-BAC from all three oncogenic strains collectively. Estimates of the non-synonymous to synonymous substitution ratio (omega) indicate that CVI988 ORFs are generally under purifying selection (omega<1), whereas UL39, UL49, UL50, RLORF6 and RLORF7 (Meq) appear to evolve under relaxed selective constraints. No CVI988 ORF was found to be under positive evolutionary selection (omega>>1).
Marek’s disease virus (MDV) is an oncogenic herpesvirus that induces fatal T cell lymphomas in chickens. With more than 20 billion doses of vaccine used annually, vaccination constitutes the cornerstone of Marek’s disease control. Despite the success of vaccination, evolution of virulence among MDV strains continues to threaten the effectiveness of the current Marek’s disease vaccines. MDV-encoded protein MEQ (MDV EcoRI Q) probably acts as a transcription factor and is considered to be the major MDV oncoprotein. MEQ sequence shows a Pro-Leu-Asp-Leu-Ser (PLDLS) motif known to bind C-terminal-binding protein (CtBP), a highly conserved cellular transcriptional corepressor with roles in the regulation of development, proliferation, and apoptosis. Here we show that MEQ can physically and functionally interact with CtBP through this motif and that this interaction is critical for oncogenesis because mutations in the CtBP-interaction domain completely abolished oncogenicity. This direct role for MEQ–CtBP interaction in MDV oncogenicity highlights the convergent evolution of molecular mechanisms of neoplastic transformation by herpesviruses because Epstein–Barr virus oncoproteins EBNA 3A and 3C also interact with CtBP. We also demonstrate that the nononcogenic MDV generated by mutagenesis of the CtBP-interaction domain of MEQ has the potential to be an improved vaccine against virulent MDV infection. Engineering MDV with precisely defined attenuating mutations, therefore, represents an effective strategy for generating new vaccines against this major poultry disease.
We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.
Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.
Abstract Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40- cre cassette within the lox P sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo . Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.
Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.
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