Michael J. Imperiale

The role of polyomaviruses in human disease

The human polyomaviruses, BK virus and JC virus, have long been associated with serious diseases including polyomavirus nephropathy and progressive multifocal leukoencephalopathy. Both viruses establish ubiquitous, persistent infections in healthy individuals. Reactivation can occur when the immune system is impaired, leading to disease progression. Recently, the human polyomavirus family has expanded with the identification of three new viruses (KI, WU and Merkel cell polyomavirus), all of which may prove to be involved in human disease. This review describes the general aspects of human polyomavirus infections and pathogenicity. Current topics of investigation and future directions in the field are also discussed.

Taxonomical developments in the family Polyomaviridae

The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV) has recommended several taxonomical revisions, as follows: The family Polyomaviridae, which is currently constituted as a single genus (Polyomavirus), will be comprised of three genera: two containing mammalian viruses and one containing avian viruses. The two mammalian genera will be designated Orthopolyomavirus and Wukipolyomavirus, and the avian genus will be named Avipolyomavirus. These genera will be created by the redistribution of species from the current single genus (Polyomavirus) and by the inclusion of several new species. In addition, the names of several species will be changed to reflect current usage.

Identification of Gangliosides GD1b and GT1b as Receptors for BK Virus

ABSTRACT Gangliosides have been shown to be plasma membrane receptors for both murine polyomavirus and SV40, while JC virus uses serotonin receptors. In contrast, little is known of the membrane receptor and entry pathway for BK virus (BKV), which can cause severe disease in immunosuppressed bone marrow and renal transplant patients. Using sucrose flotation assays, we investigated BKV binding to and interaction with human erythrocyte membranes and determined that this interaction was dependent on a neuraminidase-sensitive, proteinase K-resistant molecule. BKV was found to interact with the gangliosides GT1b and GD1b. The terminal α2-8-linked disialic acid motif, present in both of these gangliosides, is likely to be important for this interaction. We also determined that the addition of GD1b and GT1b to LNCaP cells, which are normally resistant to BKV infection, made them susceptible to the virus. In addition, BKV interacted with membranes extracted from the endoplasmic reticulum (ER) and infection was blocked by the addition of brefeldin A, which interferes with transport from the ER to the Golgi apparatus. These data demonstrate that BKV uses the gangliosides GT1b and GD1b as receptors and passes through the ER on the way to the nucleus.

BK virus and human cancer: Innocent until proven guilty

BK virus (BKV) is a polyomavirus that ubiquitously infects the human population. Following a typically subclinical primary infection, BKV establishes a life-long persistent infection in the kidney and urinary tract. BKV is known to reactivate and cause severe disease in immunosuppressed patients, particularly renal and bone marrow transplant patients. Infection of BKV in rodent animal models or cells in culture often results in tumor formation or transformation, respectively. When co-expressed with activated oncogenes, BKV large tumor antigen drives the transformation of primary human cells. An etiological role of BKV in human cancer, however, remains controversial. Multiple reports have demonstrated conflicting results in regards to the presence of BKV sequences and/or proteins in various tumor types. This review compiles the most recent findings of BKV detection in a number of human cancers. Due to the lack of conclusive causality data from these studies, there does not appear to be a definitive association between BKV and human cancers.

Role for the Adenovirus IVa2 Protein in Packaging of Viral DNA

ABSTRACT Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant,pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.

Early Events during BK Virus Entry and Disassembly

ABSTRACT BK virus (BKV) is a nonenveloped, ubiquitous human polyomavirus that establishes a persistent infection in healthy individuals. It can be reactivated, however, in immunosuppressed patients and cause severe diseases, including polyomavirus nephropathy. The entry and disassembly mechanisms of BKV are not well defined. In this report, we characterized several early events during BKV infection in primary human renal proximal tubule epithelial (RPTE) cells, which are natural host cells for BKV. Our results demonstrate that BKV infection in RPTE cells involves an acidic environment relatively early during entry, followed by transport along the microtubule network to reach the endoplasmic reticulum (ER). A distinct disulfide bond isomerization and cleavage pattern of the major capsid protein VP1 was observed, which was also influenced by alterations in pH and disruption of trafficking to the ER. A dominant negative form of Derlin-1, an ER protein required for retro-translocation of certain misfolded proteins, inhibited BKV infection. Consistent with this, we detected an interaction between Derlin-1 and VP1. Finally, we show that proteasome function is also linked to BKV infection and capsid rearrangement. These results indicate that BKV early entry and disassembly are highly regulated processes involving multiple cellular components.

Interaction of the Adenovirus IVa2 Protein with Viral Packaging Sequences

ABSTRACT We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the others known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.

Requirement of the Adenovirus IVa2 Protein for Virus Assembly

ABSTRACT The adenovirus L1 52/55-kDa protein is required for viral DNA packaging and interacts with the viral IVa2 protein, which binds to the viral packaging sequence. Previous reports suggest that the IVa2 protein plays a role in viral DNA packaging and that this function of the IVa2 protein is serotype specific. To further examine the function of the IVa2 protein in viral DNA packaging, a mutant virus that does not express the IVa2 protein was constructed by introducing two stop codons at the beginning of the IVa2 open reading frame in a full-length bacterial clone of adenovirus type 5. The mutant virus, pm8002, was defective for growth in 293 cells, although it replicated its DNA and produced early and late viral proteins. Electron microscopic and gradient analyses revealed that the mutant virus did not assemble any viral particles in 293 cells. In 293-IVa2 cells, which express the IVa2 protein, infectious viruses were produced, although the titer of the mutant virus was lower than that of the wild-type virus, indicating that these cells may not fully complement the mutation. The mutant viral particles produced in 293-IVa2 cells were heterogeneous in size and shape, less stable, and did not traffic efficiently to the nucleus. Marker rescue experiments with a wild-type IVa2 DNA fragment confirmed that the only mutations present in pm8002 were in the IVa2 gene. The results indicate that the IVa2 protein is required for adenovirus assembly and suggest that virus particles may be assembled around the DNA rather than DNA being packaged into preformed capsids.

Novel mechanisms of E2F induction by BK virus large-T antigen: requirement of both the pRb-binding and the J domains.

ABSTRACT E2F activity is regulated in part by the retinoblastoma family of tumor suppressor proteins. Viral oncoproteins, such as simian virus 40 (SV40) large-T antigen (TAg), adenovirus E1A, and human papillomavirus E7, can disrupt the regulation of cellular proliferation by binding to pRb family members and dissociating E2F-pRb family protein complexes. BK virus (BKV), which infects a large percentage of the human population and has been associated with a variety of human tumors, encodes a TAg homologous to SV40 TAg. It has been shown that BKV TAg, when expressed at low levels, does not detectably bind to pRb family members, yet it induces a serum-independent phenotype and causes a decrease in the overall levels of pRb family proteins. The experiments presented in this report show that, despite the lack of TAg-pRb interactions, BKV TAg can induce transcriptionally active E2F and that this induction does in fact require an intact pRb-binding domain as well as an intact J domain. In addition, E2F-pRb family member complexes can be detected in both BKV and SV40 TAg-expressing cells. These results suggest the presence of alternate cellular mechanisms for the release of E2F in addition to the well-established model for TAg-pRb interactions. These results also emphasize a role for BKV TAg in the deregulation of cellular proliferation, which may ultimately contribute to neoplasia.

A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting

A novel peptide with the binding characteristics of tetanus toxin was identified with phage display, for application in therapeutic protein and vector motor and sensory neuron targeting. A 12mer phage library was biopanned on trisialoganglioside (G(T1b)) and eluted with the tetanus toxin C fragment (rTTC). Phage ELISAs revealed increases in G(T1b) binding for the Tet1 and Tet2 phage clones when compared to peptideless phage (PLP). rTTC displaced both Tet1 and Tet2 phage clones from G(T1b), and both clones reduced rTTC-G(T1b) binding. Comparison of Tet1, Tet2, PLP, and the random phage library binding to PC12 and HEK293 cells revealed enhanced cellular binding by Tet1 and Tet2 phage. Tet1 phage binding was selective for neurons. Immunofluorescence also confirmed selective PC12 binding of Tet1 and Tet2 phage. Fluorescein-conjugated synthetic Tet1, but not Tet2, peptide showed strong binding to cultured PC12, primary motor neurons, and dorsal root ganglion (DRG) cells. Synthetic Tet1 bound DRG and motor neurons but not muscle in tissue sections. The enhanced neuronal binding affinity and specificity of Tet1, a novel 12 amino acid peptide, suggests potential utility for targeting neurotherapeutic proteins and viral vectors in the treatment of motor neuron disease, neuropathy, and pain.