Infectious Entry and Neutralization of Pathogenic JC Polyomaviruses.

Progressive multifocal leukoencephalopathy (PML) is a lethal brain disease caused by uncontrolled replication of JC polyomavirus (JCV). JCV strains recovered from the brains of PML patients carry mutations that prevent the engagement of sialylated glycans, which are thought to serve as receptors for the infectious entry of wild-type JCV. In this report, we show that non-sialylated glycosaminoglycans (GAGs) can serve as alternative attachment receptors for the infectious entry of both wild-type and PML mutant JCV strains. After GAG-mediated attachment, PML mutant strains engage non-sialylated non-GAG co-receptor glycans, such as asialo-GM1. JCV-neutralizing monoclonal antibodies isolated from patients who recovered from PML appear to block infection by preventing the docking of post-attachment co-receptor glycans in an apical pocket of the JCV major capsid protein. Identification of the GAG-dependent/sialylated glycan-independent alternative entry pathway should facilitate the development of infection inhibitors, including recombinant neutralizing antibodies.

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9.

Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

Targeting the primary tumor to generate CTL for the effective eradication of spontaneous metastases.

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104L(d) that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.

Direct evidence that IFN-beta functions as a tumor-suppressor protein.

Interferon-beta (IFN-beta) induces aberrant cell cycle progression as well as cytotoxicity and apoptosis. However, the relationship between the cell cycle alteration and the induction of cytotoxicity/apoptosis is unknown. Here, we report the first demonstration that the IFN-beta-induced direct cytotoxic/apoptotic effect can be separated functionally from its cell cycle effect. By using lentiviral transduction, we generated human tumor cells that stably expressed IFN-beta and were resistant to its direct cytotoxic/apoptotic effect. Despite this resistance to apoptosis, these cells showed significant S phase accumulation as measured by both FACS analyses and bromodeoxyuridine (BrdU) incorporation. Although the cells proliferated in the presence of high levels of IFN-beta, they had lost their tumorigenicity in mice. A portion of these cells was observed to undergo a tumor cell-specific senescence. Therefore, our study revealed a direct tumor-suppressor function of IFN-beta. This tumor-suppressor function was independent of IFN-beta-induced direct cytotoxic effect. It was also distinct from the IFN-beta-induced immunologic antitumor response, an indirect effect of IFN-beta. We conclude that the antiproliferative effect on human tumor cells is the collective activities of the direct cytotoxic/apoptotic effect, the cell cycle alteration that occurs as predominantly S phase accumulation, and less frequently other cell cycle effects, such as G(1) arrest, and the promotion of tumor cells into a senescent-like state.