Takashi Komatsu

Interferon-γ induced type I nitric oxide synthase activity inhibits viral replication in neurons

Abstract Type I NOS expression increases in OB neurons during VSV infection. Immunocytochemical staining of NB41A3 cells indicates constitutive expression of interferon (IFN)-γ receptor and type I NOS. IFN-γ treatment of NB41A3 cells increased NO production and type 1 NOS protein. In vitro replication of VSV, polio virus type 1, and Herpes Simplex virus type 1 (HSV-1) is significantly inhibited by IFN-γ induced type I NOS and antagonized by NOS inhibitors. In contrast, while IFN-γ treatment inhibited influenza and Sindbis virus replication, a different pathway(s) was involved. The isoform-selective NOS inhibitor, 7-nitroindazole (7NI) was used to treat mice, resulting in a 10-fold higher titer of virus in brain homogenates, and abrogated the recovery-promoting effect of interleukin-12 treatment. Thus, IFN-γ induced type I NOS activity may play an important role in host immunity against neurotropic viral infections.

Sex differences in susceptibility to viral infection of the central nervous system

We have characterized striking differences in recovery of male and female BALB/c and BALB/c-H-2dm2 (dm2) mice from an experimental neurotropic viral infection of the central nervous system (CNS). Following intranasal inoculation of vesicular stomatitis virus (VSV), assays of tissue homogenates from female mice produced lower viral titers. There was also a significant reduction in the spread of virus from the rostral to caudal end of the brain in female mice. Enhanced recovery by female mice of both strains in response to this viral insult correlates with increased levels of Nitric Oxide Synthase (NOS) types I, II, and III expression, an increased prevalence of reactive astrocytes, earlier and enhanced levels of expression of Major Histocompatibility Complex (MHC) class II molecules on astrocytes, endothelial and microglial cells, and increased T cell infiltration in the female BALB/c mouse. Taken together, these findings document sexual dimorphism in CNS immunity, and may provide an understanding of some of the mechanisms underlying many sex-biased diseases.

Determination of Kaposi's Sarcoma-Associated Herpesvirus C-Terminal Latency-Associated Nuclear Antigen Residues Mediating Chromosome Association and DNA Binding

ABSTRACT Kaposis sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) tethers viral terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence. The 1,162-amino-acid LANA protein contains both N- and C-terminal chromosome attachment regions. The LANA C-terminal domain self-associates to specifically bind TR DNA and mitotic chromosomes. Here, we used alanine scanning substitutions spanning residues 1023 to 1145 to investigate LANA self-association, DNA binding, and C-terminal chromosome association. No residues were essential for LANA oligomerization, as assayed by coimmunoprecipitation experiments, consistent with redundant roles for amino acids in self-association. Different subsets of amino acids were important for DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect DNA and chromosome binding. The DNA binding domains of LANA and EBNA1 are predicted to be structurally homologous; certain LANA residues important for DNA binding correspond to those with roles in EBNA1 DNA binding, providing genetic support for at least partial structural homology. In contrast to the essential role of N-terminal LANA chromosome targeting residues in DNA replication, deficient C-terminal chromosome association did not reduce LANA-mediated DNA replication.

Definition of Sequence Requirements for Latency-Associated Nuclear Antigen 1 Binding to Kaposi's Sarcoma-Associated Herpesvirus DNA

ABSTRACT In latent infection, Kaposis sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 (6CCC8) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since 6CCC8 is in the 5′ half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.

The Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen

Kaposis sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is associated with Kaposis sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castlemans disease (6,7,10). While the vast majority of tumor cells from these malignancies are latently infected, only a small subset of viral genes are actually expressed (5,40,47). Of these genes, the latency-associated nuclear antigen (LANA-1, LNA, or LNA1) is the only protein consistently shown to be highly expressed by in situ hybridization and immunohistochemistry (11,20,21,32). Moreover, within the past few years LANA-1 has proven to be a quite versatile protein, playing not only a pivotal role in KSHV episome persistence, but also in interacting with and influencing several cellular genes.