SUMMARY The nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed Nlrx1−/− mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1 and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1−/− mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS-activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U L 11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membranepelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membranetrafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes.During herpes simplex virus type 1 (HSV-1) assembly, over 30 different proteins come together to form three major structures: the nucleocapsid, the glycoprotein-containing envelope, and the collection of proteins located between the capsid and envelope known as the tegument (30). While it is generally accepted that capsid assembly and genome packaging occur in the nucleus, the compartment(s) in which tegument and envelope are acquired is less well defined (30). As with other herpesviruses (e.g., pseudorabies virus, varicella-zoster virus, and human cytomegalovirus), the most recent model for HSV-1 envelopment suggests that assembled nucleocapsids are shuttled out of the nucleus by a budding-fusion event on the inner and outer nuclear membranes (respectively) and then travel through the cytoplasm as unenveloped capsids until reaching a trans-Golgi network (TGN)-derived vesicle (7, 12-14, 18, 31, 40, 43, 47). While at this site, nucleocapsids are thought to acquire their final lipid bilayer in a process that also results in the acquisition of the tegument and glycoproteins. The mature virions subsequently follow the secretory pathway out to the cell surface, where they are released into the extracellular medium.Alth...

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76 gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55 gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.