SUMMARY The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. We show that expression of the essential autophagy protein Atg5 in granulocytes and macrophages is required for in vivo resistance to infection with L. monocytogenes and T. gondii. In primary macrophages, Atg5 was not required for IFNγ/LPS-mediated transcription, induction of nitric oxide, or inhibition of T. gondii replication. However, Atg5 was required for IFNγ/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect autophagosomes enveloping T. gondii, Atg5 was required for recruitment of the IFNγ-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

Human norovirus (HuNoV) is the major cause of acute nonbacterial gastroenteritis worldwide but has no clear animal reservoir. HuNoV can persist after the resolution of symptoms, and this persistence may be essential for viral maintenance within the population. Many strains of the related murine norovirus (MNV) also persist, providing a tractable animal model for studying norovirus (NoV) persistence. We have used recombinant cDNA clones of representative persistent (CR6) and nonpersistent (CW3) strains to identify a domain within the nonstructural gene NS1/2 that is necessary and sufficient for persistence. Furthermore, we found that a single change of aspartic acid to glutamic acid in CW3 NS1/2 was sufficient for persistence. This same conservative change also caused increased growth of CW3 in the proximal colon, which we found to be a major tissue reservoir of MNV persistence, suggesting that NS1/2 determines viral tropism that is necessary for persistence. These findings represent the first identified function for NoV NS1/2 during infection and establish a novel model system for the study of enteric viral persistence.

bHuman noroviruses (HuNoVs) are the major cause of epidemic, nonbacterial gastroenteritis worldwide. Due to the lack of a tractable model system and the inability to grow HuNoVs in cell culture, factors required for the norovirus (NoV) life cycle and pathogenesis in the host remain largely unknown. The discovery of murine norovirus (MNV) and the development of reversegenetics systems for this virus provide an opportunity to study these aspects of NoV infection in vitro and in vivo. Previous studies identified a single amino acid at residue 296 in the protruding (P) domain of the capsid protein that is responsible for determining the virulence of the MNV clone MNV1.CW1 in 12956/SvEv background STAT1-deficient (STAT1 ؊/؊ ) mice. In this report, we identified and characterized another determinant of lethality in the P domain that is necessary and sufficient to determine the replication and pathogenesis of the MNV clones MNV1.CW3 and CR6.STL1 in C57BL/6 background STAT1 ؊/؊ mice. Furthermore, we describe how the role of residue 296 in MNV virulence differs between STAT1 ؊/؊ mouse strains. We also describe potential interactions between subdomains of the P domain, as well as between other virus elements, which facilitate recovery of MNV using a reverse-genetics system.

Two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. For noroviruses, the contributions of T cells to viral clearance and vaccination have not been elucidated. We report here that both CD4 and CD8 T cells are required for efficient clearance of primary murine norovirus (MNV) infection from the intestine and intestinal lymph nodes. Further, long-lasting protective immunity was generated by oral live virus vaccination. Systemic vaccination with the MNV capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human Lordsdale virus provided partial protection. Fully effective vaccination required a broad immune response including CD4 T cells, CD8 T cells, and B cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. Perforin, but not interferon gamma, was required for clearance of MNV infection by adoptively transferred T lymphocytes from vaccinated hosts. These studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent CD4 and CD8 T cell responses.

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.Obesity has become a worldwide health issue, affecting children and adults in developed and emerging countries (1-3). Obesity is a disorder of both energy metabolism and appetite regulation and must be understood as a dysfunction of energy balance (3). The central nervous system (CNS) plays a critical role in the regulation of energy balance by coordinating peripheral and central signals to assess energy status and regulate feeding behavior (4 -6). While it is well known that fatty acid metabolism is an important component of the peripheral regulation of energy metabolism, recent studies indicate that neurons in the hypothalamus may monitor flux through the fatty acid synthesis pathway to ascertain energy balance (7-10).We and others have demonstrated that C75, a synthetic fatty acid synthase (FAS) 1 inhibitor (11), decreases food intake and results in profound and reversible weight loss (8,(12)(13)(14). Although inhibition of peripheral fatty acid synthesis might easily explain some of these effects, anorexia was also achieved by central (intracerebroventricular) administration of C75 (8, 14). These results suggest that the FAS pathway may play a role in energy homeostasis in the brain and that modulation of flux through this pathway in neurons or glia could alter energy levels.FAS is a lipogenic enzyme that catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids (15). In the fed state, long-chain fatty acids are synthesized and stored as triglycerides, which are broken down for...