Ken Kono

Enteroviruses, type 1 diabetes and hygiene: a complex relationship

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the hosts insulin‐producing β cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non‐obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long‐term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host‐virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed. Copyright

Group B coxsackievirus diabetogenic phenotype correlates with replication efficiency

ABSTRACT Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types. The virulent strain CVB3/28 replicated to higher titers more rapidly than CVB3/GA in the pancreas and in established beta cell cultures. Exchange of 5′-nontranslated regions between the two CVB3 strains demonstrated a variable impact on replication in beta cell cultures and suppression of in vivo replication for both strains. While any CVB strain may be able to induce T1D in prediabetic NOD mice, T1D onset is linked both to the viral replication rate and infectious dose.

Evolution of Virulence in Picornaviruses

The Picornaviridae encompass many positive-strand RNA viruses, all of which share a generally similar genome design and capsid structure, but which induce quite diverse diseases in humans and other animals. Picornavirus strains of the same serotype have been shown to express different virulence (or pathogenic) phenotypes when studied in animal models, demonstrating that key elements of pathogenesis reside in the viral genome. However, the genetics that determine the virulence phenotype of any picornavirus are poorly understood. Picornaviruses do not have virulence genes per se, but the design ofthe capsid andhow it interacts with the virus receptor expressed on the host cell surface, specific sequences within the nontranslated regions of the viral genome, as well as coding sequences that result in different protein sequences may all have a part in determining the virulence phenotype. Virulence may be better understood as a continuum from an apparent inability to induce disease to the ability to cause severe pathogenic changes. Ultimately, the ability of a picornavirus to induce disease depends upon viral genetics and how they are modulated by the host environment.

Impact of a single amino acid in the variable region 2 of the Old World monkey TRIM5α SPRY (B30.2) domain on anti-human immunodeficiency virus type 2 activity.

Variable region 1 (V1) of the SPRY domain of TRIM5alpha is a major determinant for species-specific virus restriction in primates. We previously reported that a chimeric TRIM5alpha containing baboon V1 in the background of cynomolgus monkey TRIM5alpha showed potent anti-human immunodeficiency virus type 2 (HIV-2) activity. Since baboons are reportedly sensitive to HIV-2 infection, there was a discrepancy between the ability of baboon TRIM5alpha V1 to restrict HIV-2 and baboon sensitivity to HIV-2. In the study presented here, we examined the roles of V2 and V3 of the baboon TRIM5alpha SPRY domain in its anti-HIV-2 activity. A chimeric TRIM5alpha containing the entire baboon SPRY domain showed weak anti-HIV-2 activity. This attenuation of activity was caused by a single serine-to-proline substitution in baboon TRIM5alpha V2. These findings indicate that the combination of V1 with other variable regions of SPRY is important in anti-HIV-2 activity of primate TRIM5alpha.

Abstract A16: Overexpression and ubiquitination of kinase suppressor of Ras 1 (KSR1) in human colon tumor cells

Kinase Suppressor of Ras 1 (KSR1) is required for oncogenic Ras-induced transformation in mouse embryonic fibroblasts (MEFs) and human colon tumor cells, but is dispensable for normal cellular development. It is also overexpressed in a variety of human tumors, including human colon tumor cells. Seven human colon tumor cell lines with mutated and activated Ras (GEO, CBS, FET, SW480, HCT15, DLD1, and HCT116), one with wild type Ras (CaCo2), and non-transformed human colonic epithelial cells (HCECs) were assessed for KSR1 by western blot. Compared to HCECs, all eight tumor cell lines showed a marked decrease in electrophoretic motility. Seven of eight cell lines had a clear increase in KSR1 expression regardless of Ras mutation status. The purpose of the study is to examine the mechanism by which KSR1 is up-regulated in cancer cells. Previous studies indicate that KSR proteins interact with multiple E3 ubiquitin ligase complexes including members homologous to the E6AP carboxyl terminus (HECT) domain family and the really interesting new gene (RING) family, including Cullin (Cul) 1, Cul4A, Cul4B, Cul7, damage-specific DNA binding protein 1 (DDB1), and DDB1-Cullin associating factor 1 (DCAF1), and ubiquitin protein ligase E3 component N-recognin 5 (UBR5/EDD1). Using tagged expression vectors and immunoprecipitation techniques, we investigated whether KSR1 is a substrate or a component of E3 ligase complexes. Both FLAG-tagged and endogenous KSR1 are ubiquitinated in the human colon tumor cell line, HCT-116. In HEK 293T cells, truncated KSR1 constructs were used to determine that KSR1 ubiquitination is dependent on the presence of the putative kinase domain in the C-terminal region. Furthermore, the kinase domain is sufficient for KSR1 ubiquitination by Cul4A/4B RING ligases (CRLs); however, silencing of Cul4A/4B by RNAi does not inhibit KSR1 ubiquitination. These data indicate that multiple Cullin family members may function to ubiquitinate KSR1. Additionally, KSR1 does not exclusively bind to Cullins, but also interacts with other components of the multi-subunit CRLs, including DDB1 and DCAF1. Therefore, KSR1 may not only be a direct substrate but also a member of the large complex helping to modulate activity of the CRL. Overall, these data suggest that Cul4A/4B-dependent ubiquitination of the KSR1 putative kinase may function to modulate KSR1-dependent signaling. Citation Format: Jamie L. McCall, Ken Kono, Kurt W. Fisher, Manabu Furukawa, Robert E. Lewis. Overexpression and ubiquitination of kinase suppressor of Ras 1 (KSR1) in human colon tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A16. doi: 10.1158/1557-3125.RASONC14-A16