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...

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ⌬capB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ⌬capB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ⌬capB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ⌬capB is significantly attenuated in mice; the 50% lethal dose (LD 50 ) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ⌬capB partially restores its virulence in mice. Mice immunized with LVS ⌬capB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ⌬capB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10؋ the LD 50 of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ⌬capB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.

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) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta-and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein.The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.