Inhibition of IkappaB kinase by vaccinia virus virulence factor B14

The IkappaB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-kappaB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-kappaB activation in response to TNFalpha, IL-1beta, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKbeta but not IKKalpha, suggesting the interaction occurs via IKKbeta. B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.     

Steroid hormone receptor fraction stimulation of RNA synthesis: a caution.

Permeant and impermeant labelled thiol reagents were incubated with rat liver mitochondria, and incorporation of reagent into mitochondria estimated. With permeant thiol reagents, incorporation depends on the energetic state of mitochondria; when coupled electron-transfer takes place, incorporation is fairly increased; the stimulation is abolished in the presence of an uncoupler, an electron-transfer inhibitor or inorganic phosphate. With an impermeant thiol reagent, the incorporation is unaffected by the energetic state of the mitochondria. These results favour the view of a participation of thiol groups in the energy-conserving mechanism but it cannot be ruled out that part of the unmasked thiol groups are implicated in the phosphate transport system. The observed stimulation may reflect either an increase in accessibility or in reactivity of some mitochondrial thiol groups.     

Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.     

Growth inhibition by vaccinia virus growth factor

Vaccinia virus growth factor (VGF), a highly glycosylated 77-residue epidermal growth factor (EGF)-like polypeptide encoded in vaccinia poxvirus, is reported to play an important role in stimulating growth of uninfected cells to facilitate virus infection. We have chemically synthesized the unglycosylated forms of VGF and VGF19-69, a shortened VGF analog consisting of 51 residues and comprising the EGF-homologous region (position 19-69) of VGF. Both synthetic forms of VGFs were purified to homogeneity and vigorously characterized by various criteria, including the Cf-252 ion fission fragment mass spectrometry, amino acid sequencing, and enzymatic digestion to confirm the disulfide linkages. Synthetic VGFs exhibited high affinity binding to the EGF receptors in A431, NRK 49F, NRK clone 3, and NIH 3T3 cells, but, unlike the glycosylated form, showed contrasting mitogenic activities in various cells in vitro. Synthetic VGFs showed low levels of mitogenic and colonogenic activities in NRK clone 49F cells and NIH 3T3 cells, full agonist activities in human keratinocytes and Swiss 3T3 cells, and partial agonist activities in NRK clone 3 cells. Our results suggest that the unglycosylated form of VGF is an EGF antagonist to selected cells and that the production of unglycosylated form of VGF by the cytolytic vaccinia virus may serve as a mechanism whereby inhibition of growth and metabolism of selected host cells may be used to facilitate the propagation of the virus infection.     

Activation of the vaccinia virus nicking-joining enzyme by trypsinization

The vaccinia virus nicking-joining (NJ) enzyme has been purified to homogeneity from a preparation of virus cores. The virus-specific DNA-dependent enzyme, which does not require ATP, is a single polypeptide of Mr 50,000 and possesses both endonuclease and ligase activities. The principal end product of the enzyme activity, following incubation with closed circular DNA of sufficient linking deficiency, is a linear DNA in which one of the termini has become cross-linked by the in vitro formation of a hairpin. The ability of the NJ enzyme to cross-link DNA is significantly enhanced by in vitro proteolysis. The enzymatic properties of the proteolytic digestion product, a 44-kDa polypeptide, differ in several other ways from the intact NJ enzyme. In particular, the specific activity is enhanced and the ionic strength optimum is shifted toward higher salt concentrations. It is suggested that the purified 50-kDa species is a pronuclease that is activated by proteolytic processing.     

Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.     

Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses

Vaccinia virus (VACV) encodes enzymes that cap the 5′ end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m⁷GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.     

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.