Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections.

Murine gammaherpesvirus 68 (MHV68 [also known as ) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.The herpesvirus virion is composed of an icosahedral nucleocapsid surrounded by a proteinacious layer of tegument, which in turn is enclosed by a glycoprotein-containing lipid envelope (50). The structure and protein composition of the nucleocapsid have been shown to be conserved among the three subfamilies (␣Ϫ, ␤Ϫ, and ␥Ϫ) of herpesviruses (11, 14, 62-64, 72, 74). The icosahedral nucleocapsid contains at least four integral structural proteins (the major capsid protein, triplex-1 protein, triplex-2 protein, and small capsid protein) surrounding a core of viral DNA (11,14,27,42,56,62,72,76). The other components of the virion, the envelope and the tegument in particular, are less well understood (38). The envelope contains viral glycoproteins critical for virion binding, entry, and signaling upon infection of a host cell (4,15,26,34,55,67). The tegument is the electron-dense component of the virion surrounding the capsid and interacting with the envelope (14,38,75). While the tegument component of alphaherpesviruses and betaherpesviruses is known to contain a number of gene products involved in assembly and egress of infectious virus (38) or modulation of the host cell environment upon initial infection (10,13,21,25,30,40), little is known about the protein composition of the gammaherpesvirus tegument nor about the functions of gammaherpesvirus tegument proteins immediately after infection of the cell.Study of the functions of tegument proteins in the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is hampered by the lack of cell culture systems capable of supporting productive replication of these viruses. However, murine gammaherpesvirus 68 (MHV68, or ␥HV-68) is not constrained in this manner, replicating to high titers in conventional tissue culture systems. MHV68 is a model for studying de novo gammaherpesvirus infection and pathogenesis (16,20,36,66,73). The virus is found in wild murid rodents and is capable of infecting laboratory strains of mice (8,39,48). MHV68 establishes productive infection in lung epithelia and a latent infection in splenocytes, macrophages, dendritic cells, and lung epithelial cells (23,48,57,61,69).The MHV68 virion exhibits morphological similarity to the virion organization of other gammaherpe...

The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.Virion morphogenesis among the herpesviruses is a multistep process. Nucleocapsid assembly and packaging of the viral DNA occurs in the nucleus, and nascent nucleocapsids are thought to bud into the cytoplasm in an envelopment/de-envelopment process through the nuclear inner and outer envelopes (29). The nucleocapsids are transported, in association with primary tegument proteins, through the cytoplasm to a distinct site of virion assembly. Transmission electron microscopy (TEM) studies have indicated that the major tegumentation and envelopment process occurs here, as the nascent nucleocapsids associate with electrondense tegument/glycoprotein densities conjunct to Golgi apparatus-derived vesicular membranes (29,30,46). Tegumentation results in a "wrapping" of the nascent nucleocapsid and budding into the lumen of the cytoplasmic compartments, forming nearly complete virions within the lumen. These particles then egress in a manner resembling exocytosis, with fusion of the vesicular and plasma membranes and release of virions into the extracellular space. As different tegument proteins are seemingly involved in a number of steps post-nucleocapsid assembly, identifying the role of a particular tegument protein in virion morphogenesis necessarily involves study of when and where during the egress process the protein associates with nascent virions. While the nuclear steps are thought to involve conserved tegument proteins, such ...

Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses

The gamma subfamily of herpesviruses, including EpsteinBarr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), are associated with tumors and lymphoproliferative disorders (29,43). Murine gammaherpesvirus 68 (MHV-68) is phylogenetically related to EBV and KSHV and provides major research advantages by forming plaques on cell monolayers and establishing productive lytic and latent infections in mice (10,(64)(65)(66). Unlike EBV and KSHV, MHV-68 readily establishes productive infections in many cell culture systems and thus facilitates the examination of gammaherpesvirus replication and de novo infection (66,75,76). This provides an opportunity to examine cellular responses to de novo infection and their role in regulating gammaherpesvirus activity.Prostaglandins (PGs) are potent immunoregulatory lipid mediators generated by arachidonic acid metabolism via two cellular cyclooxygenases, constitutive COX-1 and inducible 22,26,61). PGs are formed by most cell types and exert a variety of actions in various tissues and cells via PG receptors (2,16,19,33,47). PGE 2 is an important proinflammatory prostanoid that mediates many symptoms of inflammation (41,53,56,70). Production of PGE 2 is catalyzed by COX-2, which is induced in response to factors such as bacterial lipopolysaccharides, mitogens, and cytokines (17,31,57,61). However, it is unknown what role the COX-2-PGE 2 pathway might play in responding to gammaherpesvirus de novo infection.Many viruses have been linked to the modulation of COX-2 expression and PG production (9,11,25,44,46,52,60,63,(77)(78)(79). COX-2 is responsible for the exaggerated biosynthesis of PGs under acute inflammatory conditions and in a diverse group of tumors (14,23,32,67). The COX-1 and COX-2 isozymes are the pharmacologic targets of nonsteroidal antiinflammatory drugs (NSAIDs) (26,50,61,71), and NSAIDs that block COX activity and PG production have been recognized as potentially effective antiviral therapeutics (4, 6-8, 51, 62, 73, 79). However, the effect of COX inhibitors on de novo infection of a gammaherpesvirus has not been previously examined. MHV-68 has been used as a model to study the efficacy of other antiviral compounds (3, 48, 49, 68), and we used this system to address the effects of COX-2 inhibition on gammaherpesvirus replication and infection.To enhance our understanding of virus-host cell interactions involved in the replication and pathogenesis of gammaherpesviruses, we examined MHV-68 de novo infection of NIH 3T3 and BHK-21 cells as a model for analyzing the role of PG production and COX-2 activity. We compared the effects of MHV-68 and UV-irradiated MHV-68 infection on COX-2 protein expression and COX-2 promoter activation. COX inhibitors {NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide] and indomethacin} were tested for their ability to suppress MHV-68 protein expression during de novo infection. Inhibition of protein expression and virion production by NS-398 was relieved in the presence of exogenous PGE 2 ,...