BIOET, BIOT, BMMB, PATHB
Our laboratory's research interests center on the unique biology of the human polyomavirus, JC virus (JCV). JCV infects most individuals early in life and persists in their body. Infections are usually asymptomatic, but in immunocompromised hosts, JCV may cause the fatal demyelinating brain disease called progressive multifocal leukoencephalopathy (PML); ~5% of AIDS patients succumb to this disease. Recently PML has surfaced in multiple sclerosis and Crohn’s disease patients treated with natalizumab, a monoclonal antibody designed to inhibit lymphocyte migration into the tissues. Similar drugs are now on the market, elevating concerns such treatments will lead to other life-threatening events. JCV is also an oncogenic agent, inducing a variety of tumors in rodents and non-human primates. Recent reports link JCV to human cancer.
Our analysis of the JCV genome has revealed that it shares ~70% sequence identity with the genomes of the human virus BKV and the monkey virus SV40. Major sequence differences occur within transcriptional signals and the coding region for the multifunctional protein, T antigen (TAg). To determine how these sequence alterations translate into specific biological differences, our initial studies involved genetic approaches; the construction and phenotypic analysis of hybrid polyomaviruses and the use of site-directed mutagenesis to alter cis-acting replication signals and specific functional domains of T protein. These experiments began to define those sequences contributing to the restricted lytic and transforming activities of JCV. Particularly interesting were the findings that binding of the JCV TAg to the viral replication origin and to the cellular "anti-oncogene products", pRB and p53, differs with that of the corresponding SV40 protein. In addition, we have identified 3 truncated forms of TAg (T'135, T'136 and T'165) in transformed and lytically-infected cells that arise via differential splicing mechanisms. We are intrigued by the observation that T' proteins exhibit unique functions even though they are closely related at the sequence level. We have shown that JCV mutants lacking the T' proteins exhibit significantly reduced DNA replication activity. To complement our genetic approaches, the JCV TAg/T' proteins have been overproduced in eukaryotic expression systems to facilitate biochemical analyses. In addition, cell lines expressing individual JCV tumor proteins have been generated. We have found that TAg and the 3 T' proteins exhibit differential binding to the pRB family of cellular tumor suppressor proteins both in vivo and in vitro. Furthermore, this binding causes differential release of pRB-bound members of the E2F transcription factor family. Using a ras cooperation assay, we demonstrated that the JCV early proteins vary in their ability to induce transformation and immortalization of rodent cells. We have now begun to focus upon JCV’s fifth tumor protein, small t antigen (tAg), and recent data indicate tAg contributes to viral DNA replication and binds critical cellular proteins, including PP2A, p107 and p130. Important goals for our group are to identify the basis for functional differences among the closely related JCV early proteins and to understand how the alternative splicing process, which generates the five JCV early transcripts, is regulated.
Figure 1: JCV genome. The inner circle represents the 5 kb circular double-stranded DNA, the outer arrows indicate the encoded viral proteins. The early region specifies 5 regulatory proteins produced by alternative splicing of the early mRNA. The 3 T' proteins are 135, 136 and 165 amino acids in length, and share their N-terminal 132 amino acids with the multi-functional TAg, of which 81 amino acids are also shared with tAg. T'165 also shares its 33 C-terminal residues with TAg, while T'135 and T'136 have unique 3 and 4 amino acid C- termini, respectively, in a different reading frame. The late region specifies 3 proteins that comprise the capsid and a 4th protein, called agnoprotein or LP1 that regulates some aspects of viral transcription and DNA replication. The regulatory region (RR) contains numerous, overlapping cis-acting sequences, including early/late promoters and enhancer (P-E) and core/auxiliary origins of DNA replication (Ori).
Figure 2: PML Brain. Progressive multifocal leukoencephalopathy (PML) results from the lytic destruction of JCV-infected oligodendrocytes, the glial cells in the central nervous system that produce myelin. Multiple small lesions in the white mater of the brain progressively enlarged and coalesced to produce the large demyelinated (no staining) lesion observed in the diseased brain on the right. A normal brain section is shown on the left for comparison.