Cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of NF-kappaB activation.

NF-kappaB comprises a family of transcription factors that regulate key immune processes. In this study, the effects of orthopoxvirus infection upon the activation of NF-kappaB were examined. During the early phase of infection, cowpox virus can inhibit the induction of NF-kappaB-regulated gene expression by interfering with the process of IkappaBalpha degradation. Although either okadaic acid or tumor necrosis factor (TNF) treatment of infected cells can induce IkappaBalpha phosphorylation, further processing of IkappaBalpha is inhibited. These results suggest that cowpox virus is capable of inhibiting the activation of NF-kappaB at a point where multiple signal transduction pathways converge. Other orthopoxviruses affect NF-kappaB activity, but in a type-specific manner. Raccoonpox virus and vaccinia virus (Copenhagen strain) negatively affect NF-kappaB induction by TNF. In contrast, the modified vaccinia virus Ankara strain induces NF-kappaB activation, even in the absence of other stimuli. These findings suggest that orthopoxviruses may affect a broad range of virus-host interactions through their effects upon NF-kappaB activation. Moreover, because of the central role for NF-kappaB in immune processes and disease, these type-specific effects may contribute significantly to the immunogenic and pathogenic properties of poxviruses.

A 43-nucleotide RNA cis-acting element governs the site-specific formation of the 3' end of a poxvirus late mRNA.

The 3' ends of late mRNAs of the ati gene, encoding the major component of the A-type inclusions, are generated by endoribonucleolytic cleavage at a specific site in the primary transcript [Antczak et al., (1992), Proc. Natl. Acad. Sci. USA 89, 12033-12037]. In this study, sequence analysis of cDNAs of the 3' ends of ati mRNAs showed these mRNAs are 3' polyadenylated at the RNA cleavage site. This suggests that ati mRNA 3' end formation involves cleavage of a late transcript, with subsequent 3' polyadenylation of the 5' cleavage product. The RNA cis-acting element, the AX element, directing orientation-dependent formation of these mRNA 3' ends, was mapped to a 345-bp AluI-XbaI fragment. Deletion analyses of this fragment showed that the boundaries of the AX element are within -5 and +38 of the RNA cleavage site. Scanning mutagenesis showed that the AX element contains at least two subelements: subelement I, 5'-UUUAU downward arrowCCGAUAAUUC-3', containing the cleavage site ( downward arrow), separated from the downstream subelement II, 5'-AAUUUCGGAUUUGAAUGC-3', by a 10-nucleotide region, whose composition may be altered without effect on RNA 3' end formation. These features, which differ from those of other elements controlling RNA processing, suggest that the AX element is a component of a novel mechanism of RNA 3' end formation.

A third distinct tumor necrosis factor receptor of orthopoxviruses.

Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and crmC. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-alpha (LTalpha) and blocked their in vitro cytolytic activity. Secreted viral CrmD bound TNF and LTalpha and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses.

Poxvirus genomes encode a secreted, soluble protein that preferentially inhibits beta chemokine activity yet lacks sequence homology to known chemokine receptors.

Poxvirus genomes encode several proteins which inhibit specific elements of the host immune response. We show the "35K" virulence gene in variola and cowpox viruses, whose vaccinia and Shope fibroma virus equivalents are strongly conserved in sequence, actually encodes a secreted soluble protein with high-affinity binding to virtually all known beta chemokines, but only weak or no affinity to the alpha and gamma classes. The viral protein completely inhibits the biological activity of monocyte chemotactic protein-1 (MCP-1) by competitive inhibition of chemokine binding to cellular receptors. As all beta chemokines are also shown to cross-compete with MCP1 binding to the viral protein, we conclude that this viral chemokine inhibitor (vCCI) not only interacts through a common binding site, but is likely a potent general inhibitor of beta chemokine activity. Unlike many poxvirus virulence genes to date, which are clearly altered forms of acquired cellular genes of the vertebrate immune system, this viral chemokine inhibitor (vCCI) shares no sequence homology with known proteins, including known cellular chemokine receptors, all of which are multiple membrane-spanning proteins. Thus, vCCI presumably has no cellular analogue and instead may be the product of unrelenting sequence variations which gave rise to a completely new protein with similar binding properties to native chemokine receptors. The proposed function of vCCI is inhibition of the proinflammatory (antiviral) activities of beta chemokines.

Cowpox virus genome encodes a second soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF but not LT alpha.

We show the cowpox genome (Brighton Red strain) contains a single copy gene, crmC, expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The approximately 25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LT alpha, TNF beta) and lacks the conserved (but nonhomologous) approximately 150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF.

Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B.

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.

The mode of death of pig kidney cells infected with cowpox virus is governed by the expression of the crmA gene.

Pig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing the crmA gene; recombinant cowpox virus A602, lacking the crmA gene; or cowpox virus A604, a revertant of virus A602, expressing the crmA gene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that the crmA gene is necessary to inhibit processes of apoptosis induced in LLC-PK1 cells by infection with cowpox virus. Thus in cells of certain types, the crmA gene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replication in vivo, potentially facilitating both virus trafficking and interference with antiviral immune defenses.

Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A.

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 x 10(5) M-1 s-1, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1 beta-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.

Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.

Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.

Cowpox virus contains two copies of an early gene encoding a soluble secreted form of the type II TNF receptor.

The inverted terminal repeats of the DNA of cowpox virus (Brighton Red strain) contain the crmB gene, an additional member of a family of viral genes that modify cytokine responses to infection. The crmB gene is transcribed from an early promoter. The primary product is a 355-amino-acid protein containing a signal peptide sequence and three potential N-linked glycosylation sites. The mature gene product is a secreted soluble protein that has an apparent molecular mass of 48 kDa. TNF alpha and TNF beta bind to this protein in a competitive manner, consistent with the sequence of its N-terminal 176 amino acids, which closely resembles the ligand-binding domains of the type II (75-kDa) human TNF receptor. The sequence of the C-terminal 161 amino acids of the CrmB protein is unlike that of human TNF receptors, but overall, the CrmB protein is similar to the T2 proteins of the leporipoxviruses (48% identity) and the predicted product of the G4R/G2R open reading frame of variola virus (85% identity), suggesting that not only the TNF-binding domains but also the C-terminal regions contribute to the functions of these viral proteins. These results show that orthopoxiviruses such as cowpox virus encode secreted forms of TNF receptors that can contribute to the modification of TNF-mediated antiviral processes.

Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition.

We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.

Poxviral modifiers of cytokine responses to infection.

Poxviruses include some of the most virulent of all human pathogens. In part, the virulence of these viruses stems from their abilities to counter host defenses against infection. A family of cytokine-response modifiers encoded by the poxviruses contribute to these countermeasures. The poxviral cytokine-response modifiers appear to affect cytokine responses in at least four different ways: (a) by inhibiting the synthesis and release of cytokines from infected cells; (b) by interfering with the interaction between a cytokine and its receptor; (c) by inhibiting cytokine signal transmission; and (d) by synthesizing virus-encoded cytokines that antagonize the effects of host cytokines mediating antiviral processes. Known poxviral, cytokine-response modifiers include CrmA, an inhibitor of the interleukin-1 beta converting enzyme; several secreted soluble receptors for tumor necrosis factor, interleukin-1, and interferon-gamma; and poxvirus-encoded growth factors resembling epidermal growth factor. Collectively, these and other as yet unidentified cytokine-response modifiers contribute to the inhibition of a variety of nonspecific and virus-specific immune defenses against virus infection. Information gained on the mechanisms used by poxviruses to modify cytokine-mediated processes should assist the development of novel therapies for a variety of diseases.

Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA.

The cowpox virus late mRNAs encoding the major protein of the A-type inclusions have 3' ends corresponding to a single site in the DNA template. The DNA sequence of the Alu I-Xba I fragment at this position encodes an RNA cis-acting signal, designated the AX element, which directs this RNA 3' end formation. In cells infected with vaccinia virus the AX element functions independently of either the nature of the promoter element or the RNA polymerase responsible for generating the primary RNA. At late times during virus replication, vaccinia virus induces or activates a site-specific endoribonuclease that cleaves primary RNAs within the AX element. The 3' end produced by RNA cleavage is then polyadenylylated to form the 3' end of the mature mRNA. Therefore, the poxviruses employ at least two mechanisms of RNA 3' end formation during the viral replication cycle. One mechanism, which is operative at early times in viral replication, involves the termination of transcription [Rohrmann, G., Yuen, L. & Moss, B. (1986) Cell 46, 1029-1035]. A second mechanism, which is operative at late times during viral replication, involves the site-specific cleavage of primary RNAs.

Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein.

Supernatants from vaccinia virus (VV)-infected CV-1 cells were examined and found to contain a 33 kd protein capable of binding murine interleukin-1 beta (mIL-1 beta). A VV open reading frame (ORF) that exhibits 30% amino acid identity to the type II IL-1 receptor was expressed in CV-1-EBNA cells and shown specifically to bind mIL-1 beta. A similar ORF from cowpox virus was expressed and also specifically bound mIL-1 beta. A recombinant VV was constructed in which this ORF was disrupted (vB15RKO). Supernatants from vB15RKO-infected cells did not contain an IL-1-binding protein. Supernatants from VV-infected CV-1 cells were capable of inhibiting IL-1-induced murine lymphocyte proliferation in vitro while supernatants from vB15RKO infected cells did not. Intracranial inoculation of mice with vB15RKO suggests that this ORF is involved in VV virulence. The possible role of a virus-encoded IL-1-binding protein in the pathology of a poxvirus infection and its relationship to other poxvirus-encoded immune modulators is discussed.

The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication.

The ability of vaccinia virus to inhibit processes of cap-dependent translational initiation by inactivating the eukaryotic translation initiation factor 4E (eIF-4E) has been examined. Analyses of the quantities of eIF-4E present in either uninfected mouse L929 cells or vaccinia virus-infected cells showed that during the first 12 hr of virus replication, when there is a marked decrease in host gene expression in infected cells, there is no change in the total amount of eIF-4E present. Analyses of eIF-4E that was metabolically labeled with [32P] and then purified by affinity chromatography using m7GTP-Sepharose 4B, indicated that neither the incorporation of radiolabel into eIF-4E nor the amounts of eIF-4E capable of binding to cap structures changed significantly during virus replication. Immunodetection of phosphorylated and unphosphorylated eIF-4E in cell lysates fractionated by two-dimensional gel electrophoresis showed that the steady-state levels of phosphorylated and unphosphorylated forms of eIF-4E were similar in uninfected and virus-infected cells. These results suggest that vaccinia virus does not gain preferential translation of viral mRNAs over other mRNAs in the cell by reducing either eIF-4E phosphorylation or its ability to bind to the cap structure.

Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme.

Cowpox virus effectively inhibits inflammatory responses against viral infection in the chick embryo. This study demonstrates that one of the viral genes necessary for this inhibition, the crmA gene (a cytokine response modifier gene), encodes a serpin that is a specific inhibitor of the interleukin-1 beta converting enzyme. This serpin can prevent the proteolytic activation of interleukin-1 beta, thereby suppressing an interleukin-1 beta response to infection. However, the modification of this single cytokine response is not sufficient to inhibit inflammatory responses. This suggests that cowpox virus encodes several cytokine response modifiers that act together to inhibit the release of pro-inflammatory cytokines in response to infection. These viral countermeasures to host defenses against infection may contribute significantly to the pathology associated with poxvirus infections.

Transcription of the terminal loop region of vaccinia virus DNA is initiated from the telomere sequences directing DNA resolution.

The telomeres of vaccinia virus DNA are transcribed at late times after infection. Analysis of cDNAs of RNA transcripts of the terminal loop region of the viral DNA shows that both inverted and complementary forms of the terminal loop region are transcribed. These late RNAs, which contain 5' poly(A) sequences, do not appear to encode any proteins. The transcriptional start sites for most of these RNAs are within the sequences that direct the resolution of concatemeric DNA replication intermediates (M. Merchlinsky and B. Moss, 1989, J. Virol. 63, 4354-4361). This suggests that the process of DNA resolution may involve transcription initiated from the telomere sequences required for resolution.

Transcription of orthopoxvirus telomeres at late times during infection.

The telomeres of orthopoxvirus DNAs consists largely of short repeated sequences organized into at least two separate sets. Although the sequence composition of the orthopoxvirus telomeres is highly conserved, these regions do not appear to encode any proteins. At late times during infection, the telomeres of vaccinia virus are transcribed. A promoter in the region between the two sets of repeats directs transcription towards the hairpin-loop end of the viral DNA. This promoter resembles the promoters of other poxvirus late genes, and directs the synthesis of RNAs whose structure is consistent with the presence of 5' poly(A) sequences typical of late RNAs. The lengths of these late transcripts suggest that some transcription extends through the hairpin-loop region. This might occur either when the genome is in a monomeric form or when the genome is in the concatemeric form of the DNA replication intermediate. The function of late transcription of the telomeres is unclear, but similar transcription of the telomeres of vaccinia virus, cowpox virus, and raccoonpox virus suggests that such transcription may have a role in viral replication.

Vaccinia virus directs the synthesis of early mRNAs containing 5' poly(A) sequences.

mRNAs transcribed from late promoters of several poxvirus genes contain 5' poly(A) sequences that are not complementary to the viral DNA. In contrast, early mRNAs containing 5' poly(A) sequences have not previously been identified. Modifications to the sequence of the promoter of an early gene of cowpox virus enable this promoter to direct the synthesis of RNAs containing 5' poly(A) sequences. When the sequence 3'-ATTTA-5', which is present at the RNA start-sites of several late promoters, is positioned such that the RNA start-site of the early promoter is at the first thymidylate in this sequence, this early promoter directs the synthesis of early RNAs containing 4-11 adenylates at their 5' ends. When two of the thymidylates in the sequence 3'-ATTTA-5' are removed, the promoter directs the synthesis of early RNAs lacking 5' poly(A) sequences. These results are consistent with the proposal that 5' polyadenylylation of poxvirus RNAs occurs by repetitive transcription of thymidylates in the sequence 3'-ATTTA-5' often present at the sites of transcriptional initiation. In addition, these results demonstrate that 5' polyadenylylation of viral RNAs is not exclusively a late function. The promoter regions of a few early genes of vaccinia virus contain the sequence 3'-ATTTA-5'. Analyses of the transcripts of one of these genes, the D5 gene, indicated that these mRNAs contain 5' poly(A) sequences, suggesting that early mRNAs of a subset of viral genes contain 5' poly(A) sequences.

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase.

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.