Role of the Leucine Zipper of Marek's Disease Virus Oncoprotein Meq in Pathogenesis

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. The MDV genome encodes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to c-Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. Previous in vitro data has suggested that Meq homodimers may be involved in regulating viral latency/reactivation, while Meq/c-Jun heterodimers are involved in transformation. Therefore, this research investigates the role of Meq homodimers and Meq-Jun heterodimers in the pathogenicity of MDV, by generating chimeric meq genes, which contain the leucine zipper region of the yeast transcription factor GCN4 (meqGCN) or leucine zipper region of c-Fos (meqFos). Thus producing Meq proteins that exclusively homodimerize (MeqGCN) or heterodimerize with Jun family members (MeqFos). Recombinant viruses (rMd5-MeqGCN and rMd5- MeqFos) containing the chimeric genes meqGCN or meqFos, respectively, in place of parental meq were generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The chimeric genes were evaluated in vitro and retained DNA binding and transactivation/repressive functions, however, selected cells expressing MeqGCN and MeqFos had reduced colony formation in soft agar. Both the rMd5-MeqGCN and rMd5- MeqFos viruses replicated in vitro and in vivo, but rMd5-MeqGCN was unable to transform T-cells in infected chickens, while rMd5-MeqFos induced preneoplastic nerve lesions in 50% of infected birds. However, a third virus rMd5-MeqFos/GCN, which contains one copy of each meqGCN and meqFos, induced preneoplastic nerve lesions in 60% of infected chickens and neoplastic lesions in 20% of infected chickens. These data provide the first in vivo evidence that both Meq homodimers and heterodimers are necessary for MDV induced transformation

Use of Avian Bornavirus Isolates to Induce Proventricular Dilatation Disease in Conures

The fulfillment of Koch’s postulates shows that the virus causes proventricular dilatation disease in parrots

Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens ▿

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation

Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture

<div><p>Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.</p></div

Anti-viral activity of Ribavirin on PaBV-4 in duck embryonic fibroblasts (DEF).

<p>(A) PaBV-4 positive cell foci, assessed by indirect immunocytochemistry assay. (B) Percent area of infected cells, assessed by indirect immunocytochemistry assay. DEF infected with 64 ffu (black circle), 7 ffu of PaBV-4 (grey square), or no virus (white triangle), incubated with ribavirin for 5 days, and then incubated a further 5 days in the absence of ribavirin. Data presents as median ± 25^th and 75^th percentile. *, compared with no added ribavirin (<i>P</i> <0.05, ANOVA on ranks with Tukey test).</p

Time dependency to reduce viral load.

<p>(A) PaBV-4 positive cell foci, assessed by indirect immunocytochemistry assay. (B) Percent area of infected cells, assessed by indirect immunocytochemistry assay. DEF infected with 64 ffu (black circle), 7 ffu of PaBV-4 (grey square), or no virus (white triangle), incubated for 0, 1, 2, or 5 days with 25μg/mL ribavirin. Data presents as median ± 25^th and 75^th percentile.</p

Guanosine inhibition of ribavirin antiviral activity on PaBV-4.

<p>(A) qRT-PCR results reported as mean (±SD) relative change in P gene mRNA expression, compared to control group (neither guanosine nor ribavirin added). *, compared with no guanosine within ribavirin treatment group (<i>P</i> <0.05, ANOVA with Holm-Sidak method). (B) Indirect immunocytochemistry results presented as median ± 25^th and 75^th percentiles positive stained cell foci. (C) Indirect immunocytochemistry results presented as median ± 25^th and 75^th percentiles of the percentage area of infected cells.</p

Potential anti-viral synergism of baicalein and ribavirin on PaBV-4.

<p>Duck embryonic fibroblasts infected with 7 (white hashed bars) or 64 (grey bars) ffu PaBV-4, incubated with 0.05 μg/mL baicalein, 5.0 μg/mL ribavirin, both drugs or neither. Cells were analyzed by qRT-PCR with results (mean ±SD) shown as the fold change in P gene mRNA expression relative to the expression of the groups treated with neither drug.</p

Anti-viral activity of Ribavirin on PaBV-4 in duck embryonic fibroblasts (DEF).

<p>(A) PaBV-4 positive cell foci, assessed by indirect immunocytochemistry assay. (B) Percent area of infected cells, assessed by indirect immunocytochemistry assay. DEF infected with 64 ffu (black circle), 7 ffu of PaBV-4 (grey square), or no virus (white triangle), incubated with ribavirin for 5 days, and then incubated a further 5 days in the absence of ribavirin. Data presents as median ± 25^th and 75^th percentile. *, compared with no added ribavirin (<i>P</i> <0.05, ANOVA on ranks with Tukey test).</p