We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

Summary Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol, and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

Nasopharyngeal carcinoma, Kaposi's sarcoma, and B-cell lymphomas are human malignancies associated with gammaherpesvirus infections. Members of this virus family are characterized by their ability to establish latent infections in lymphocytes. The latent viral genome expresses very few gene products. The infected cells are therefore poorly recognized by the host immune system, allowing the virus to persist for long periods of time. We sought to identify the cell-specific factors that allow these viruses to redirect their life cycle from productive replication to latency. We find that the cellular transcription factor NF-B can regulate this process. Epithelial cells and fibroblasts support active (lytic) gammaherpesvirus replication and have low NF-B activity. However, overexpression of NF-B in these cells inhibits the replication of the gammaherpesvirus murine herpesvirus 68 (MHV68). In addition, overexpression of NF-B inhibits the activation of lytic promoters from MHV68 and human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). In lymphocytes latently infected with KSHV or EBV, the level of NF-B activity is high, and treatment of these cells with an NF-B inhibitor leads to lytic protein synthesis consistent with virus reactivation. These results suggest that high levels of NF-B can inhibit gammaherpesvirus lytic replication and may therefore contribute to the establishment and maintenance of viral latency in lymphocytes. They also suggest that NF-B may be a novel target for the disruption of virus latency and therefore the treatment of gammaherpesvirus-related malignancies.

␥-Herpesviruses, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are important human pathogens, because they are involved in tumor development. Murine ␥-herpesvirus-68 (MHV-68 or ␥HV-68) has emerged as a small animal model system for the study of ␥-herpesvirus pathogenesis and host-virus interactions. To identify the genes required for viral replication in vitro and in vivo, we generated 1,152 mutants using signature-tagged transposon mutagenesis on an infectious bacterial artificial chromosome of MHV-68. Almost every ORF was mutated by random insertion. For each ORF, a mutant with an insertion proximal to the N terminus of each ORF was examined for the ability to grow in fibroblasts. Our results indicate that 41 genes are essential for in vitro growth, whereas 26 are nonessential and 6 attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF 54 being important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole genome level validates signature-tagged transposon mutagenesis screening as an effective genetic system to identify important virulent genes in vivo and define interactions with the host immune system. functional mapping ͉ herpesvirus ͉ bacterial artificial chromosome ͉ deoxyuridine-triphosphatase ͉ transposition

Murine gammaherpesvirus 68 (MHV-68 [also referred to as ␥HV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV).Gammaherpesviruses are known to establish latency in lymphocytes and are associated with tumorigenesis (5-7, 10, 48). Two important human pathogens in the gammaherpesvirus subfamily of herpesviruses are Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and EpsteinBarr virus (EBV). KSHV and EBV are associated with several malignancies, including B-cell lymphomas, nasopharyngeal carcinoma, and Kaposi's sarcoma (22,23,27,30,32). Studies of KSHV and EBV are limited by the lack of cell lines able to support efficient productive infection and by the restricted host ranges of the viruses (11, 33). Murine gammaherpesvirus 68 (MHV-68 [also referred to as ␥HV68]) is another member of the gammaherpesvirus subfamily. However, in vitro cell culture systems are available to study productive de novo infection by MHV-68, as well as latency and reactivation (34, 40). MHV-68 forms plaques on monolayers of many cell lines, making it possible to genetically manipulate the viral genome. MHV-68 establishes lytic and latent infections in laboratory mice (47), providing a system for examining host-virus interactions (24,25,36,42,43). These characteristics of MHV-68 make it possible to examine the functions of individual viral genes at various points during the viral life cycle, including de novo infection. De novo infection analyses have not been possible for other gammaherpesviruses such as EBV and KSHV.Herpesviruses have two distinct life cycle phases, latency and lytic replication. Reactivation from latency to lytic replication is essential for transmission of the virus from host to host and thus is one important aspect of herpesvirus biology. A viral protein, replication and transcription activator (RTA) is primarily encoded by open reading frame (ORF) 50, which is well conserved among gammaherpesviruses. RTA is necessary and sufficient to reactivate MHV-68 and drive the lytic cycle to completion in latently infected B cells (14,19,54,55). Similarly, KSHV RTA has been shown to be sufficient to reactivate the virus from latently infected B cells derived from KSHVassociated lymphomas (20,46). Although two EBV proteins, RTA and ZEBRA, function in a cooperative manner to reactivate the viral lytic cycle (3, 18, 21), RTA alone can disrupt latency in some latently infected cell lines (31, 56). These studies indicate that RTA of gammaherpesviruses plays a conserved role in virus reactivation.We have constructed custom membrane arrays representing nearly all of the known and predicted MHV-68 ORFs to explore the patterns of viral gene expression. To illustrate the value of genome-wide transcription analysis, we used the MHV-68 DNA arrays to identify a novel regulatory element for a specific gene, to identify latency-associated transcripts not previously recognized, and to define the genome-wide effects of a specific gene...

Summary A conserved herpesviral kinase has been shown to play multiple vital roles in the life cycle of herpesviruses. ORF36, the kinase of murine gamma-herpesvirus 68 (MHV-68), was identified to counteract antiviral type I interferon (IFN) response through the screening of mutant viruses. ORF36 binds to activated interferon regulatory factor 3 (IRF-3) in the nucleus and inhibits the interaction between the IRF-3 and the co-transcriptional activator CBP, thereby suppressing the recruitment of RNA polymerase II to interferon beta promoter. Although the conserved kinase activity is not absolutely required for this interaction, the anti-IFN function of ORF36 is conserved among all herpesvirus subfamilies. Mutant viruses without ORF36 induce more interferon response and are attenuated both in vitro and in vivo. Our data suggest that herpesviruses have evolved an inhibitor of antiviral IFN defense within their conserved kinase, which is critical for herpesvirus to evade host immune control and persist in a host.

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.