Background: Human polyomavirus JC (JCV) is the etiologic agent of a brain disease, known as progressive multifocal leukoencephalopathy (PML). The JCV genome encodes a small multifunctional phospho-protein, agnoprotein, from the late coding region of the virus, whose regulatory functions in viral replication cycle remain elusive. In this work, the functional role of JCV and SV40 agnoproteins in virion release was investigated using a point mutant (Pt) of each virus, where the ATG codon of agnoprotein was mutated to abrogate its expression. Results: Analysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content. Conclusions: The results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.