Virus-induced loss of class I MHC antigens from the surface of cells infected with myxoma virus and malignant rabbit fibroma virus.

Shope fibroma virus (SFV) is a leporipoxvirus that causes localized benign fibromas in immunocompetent adult rabbits that spontaneously regress due, in part, to a cell-mediated immune response. Myxoma virus (MYX) and malignant rabbit fibroma virus (MRV) are related leporipoxviruses that induce rapidly lethal generalized infections accompanied by tumors and immunosuppression. Because only these latter two viruses are known to compromise cell-mediated antiviral responses, cell surface levels of class I MHC molecules in SFV-, MRV-, and MYX-infected cells were investigated by fluorescent activated cell sorting analysis using a variety of different anti-HLA mAb. After infection with MYX or MRV there is a rapid decrease in the levels of detectable surface class I epitopes as detected by each antibody and by 24 h postinfection class I MHC Ag levels at the cell surface approach the level of background fluorescence observed with control antibodies. In contrast, only a moderate class I decrease is seen during infection with either SFV or vaccinia virus, an orthopoxvirus that is neither tumorigenic nor immunosuppressive. Surface class I marker loss induced by MYX and MRV is not simply due to nonspecific inhibition of total cellular protein synthesis by the viruses because class I levels decrease much further than the extent measured by estimating surface marker turnover in the presence of the protein synthesis inhibitor cycloheximide. Thus the loss of cellular surface class I molecules greatly exceeds the drop in level caused by complete blockage of host cell gene expression, and must involve removal or masking of preexisting class I epitopes from the cell surface by MRV/MYX. Cell surface levels of the transferrin receptor are unaffected by MYX and MRV infection, suggesting the observed class I decrease is not a nonspecific effect on total cell surface glycoproteins. Analysis of cells infected with MRV/MYX in the presence of cycloheximide or of cytosine arabinoside, an inhibitor of poxviral DNA replication, indicates that the class I marker loss is mediated in part by one or more viral late gene products. A probable explanation is that MRV/MYX late protein(s) interact with the class I MHC complex to either physically sequester these away from the cell surface and inhibit their recycling or else induce a conformational change that precludes recognition by all class I antibodies tested. In either event, we propose that such a major perturbation of the class I MHC complex would likely downregulate the class I-mediated presentation of viral Ag required to initiate cell-mediated immunity to these viruses.     

Synthesis and structure-activity study of myxoma virus growth factor

Myxoma virus growth factor (MGF) is an 85-residue peptide derived from the gene product of a DNA tumor virus that infects rabbits. The carboxyl domain of MGF possesses about 40% sequence homology with the epidermal growth factor (EGF). This EGF-like domain covering residues 30-83 was synthesized and found to possess putative activities of EGF. It was, however, about 200-fold less active than EGF in the competitive binding of EGF receptor in A431 cells and the stimulation of [3H]-thymidine uptake in NRK 49F cells. MGF(30-83) is a basic and a hydrophobic peptide rich in beta-sheet structure. These features in MGF tend to promote aggregation, leading to precipitation even in strongly denaturing solutions. Thus, the refolding of MGF was achieved with difficulty and resulted in low yield. To increase the synthetic yield of MGF(30-83), a cluster of acidic amino acids was added to the NH2-terminus of MGF(30-83). This approach was found to be effective in minimizing the refolding difficulties and allowed accessibility to the synthesis of analogues in this class of compounds. The relationships of structure and function of MGF were studied by using analogues with point substitution by the corresponding D-amino acid or by Ala at position 44 or 52 and analogues with deletion of basic residues from the amino terminus. Modifications of both the receptor contact and the structural residues greatly reduced the potency of MGF(30-83), and the overall result correlated well with the known structure-activity of the EGF family.     

M062 is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host SAMD9 in human cells

Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.     

Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophage and T-cell activation in vivo

M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25(+) infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R(+) dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.     

African swine fever virus multigene family 360 genes affect virus replication and generalization of infection in Ornithodoros porcinus ticks

Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host, Ornithodoros porcinus porcinus, an MGF360/530 gene deletion mutant (Pr4Delta35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Delta35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Delta3-C2, which lacked three MGF360 genes (3HL, 3Il, and 3LL), exhibited reduced viral growth in ticks. Pr4Delta3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Delta3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Delta3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.     

M135R is a novel cell surface virulence factor of myxoma virus

Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified.     

Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase.

The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells. Fractionation and membrane treatment studies showed that approximately 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon. Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and delta GKT174-176). The K175Q and delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a homo- or heteromultimer. An allele with a deletion of the first half of the gene, virB11 delta1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain. Products of both virB11 delta1-156 and virB11 delta158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A. tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants.     

Lessons in Détente or know thy host: The immunomodulatory gene products of myxoma virus

The poxvirus, myxoma virus, encodes within its genome at least eleven different proteins that compromise, skew, or disable the innate and adaptive responses of its hosts. In the laboratory rabbit, Oryctolagus cuniculus, these effects result in myxomatosis, a fatal condition characterized by skin lesions and systemic immunosuppression. Interestingly, while myxoma infection also causes skin lesions in its natural host and in natural populations of O. cuniculus in Australia where this novel host and the virus have co-evolved, the condition of myxomatosis does not ensue and infection is not fatal. In this review I discuss the biochemical properties of the characterized immunomodulatory proteins of myxoma virus, and their pathogenic effects in laboratory rabbits. Disruption of any one myxoma immunomodulatory gene diminishes the severity of the infection without compromising infectivity. Thus, the characterized immunomodulatory genes appear not to be required for a productive infection in vivo. The differences in the severity of their effects in laboratory-bred versus wild O. cuniculus suggest that the outcome of myxoma infection is a consequence of the interplay between the viral immunomodulatory gene products and the cells and molecules of the host immune system.     

The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells.

The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.     

The nef gene of simian immunodeficiency virus SIVmac1A11.

The role of the simian immunodeficiency virus (SIV) nef gene in viral replication was investigated in several tissue culture systems. SIVmac1A11 is a molecularly cloned virus which replicates in both peripheral blood mononuclear cells (PBMC) and macrophages, although no disease is observed in infected rhesus macaques. In this report, we demonstrate that SIVmac1A11 contains a full open reading frame for nef which specifies a 37-kDa protein. To investigate the effects of nef on viral replication, a 70-bp deletion was introduced into the nef gene of SIVmac1A11. Analysis of infected cell extracts by immunoblotting revealed that both SIVmac1A11 and nef deletion virus SIVmac1A11 delta nef produced the same viral proteins, except that Nef was absent in the mutant virus. The deletion mutation did not affect viral replication in PBMC, in monocyte-derived and alveolar macrophages obtained from rhesus macaques, and in human cell lines HUT-78 and CEMx-174. In addition, SIVmac1A11 and SIVmac1A11 delta nef exhibited similar patterns of cytopathologic changes and ultrastructural appearances in infected cells. SIVmac1A11 and SIVmac1A11 delta nef did not infect human tumor macrophage cell line U937, GCT, THP-1, or HL-60 cells, although virus was produced after these cells were transfected with either wild-type or nef mutant viral DNA. Similar levels of virus were recovered from U937 and THP-1 cells transfected with mutant and parental proviral DNAs. In transient expression assays in a T-cell line and a macrophage line, the nef protein of SIVmac1A11 did not significantly suppress or enhance expression of the chloramphenicol acetyltransferase reporter gene linked to the SIVmac long terminal repeat. Thus, abrogation of nef did not affect several in vitro properties of SIVmac1A11, including patterns of viral infection in rhesus PBMC, rhesus macrophages, or human T-cell lines.     

Serp2, an Inhibitor of the Interleukin-1β-Converting Enzyme, Is Critical in the Pathobiology of Myxoma Virus

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1β (IL-1β)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1β by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860–5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2⁻ mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4⁺ T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2⁻ mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2⁻ mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2⁻ mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1β-converting-enzyme-dependent pathways.     

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.     

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.     

Vaccinia virus F12L protein is required for actin tail formation, normal plaque size, and virulence

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (vDeltaF12L) and a revertant virus with the F12L gene reinserted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antibody was also inserted into the F12L locus of vDeltaF12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99.5%) reduction in actin tail formation, and the levels of cell-associated enveloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, vDeltaF12L produced a very small plaque. The vDeltaF12L virus was severely attenuated in vivo, such that a dose of vDeltaF12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.     

The effect of buprenorphine on the course of disease in laboratory rabbits infected with myxoma virus

The only method of assessing the virulence of myxoma virus is to record survival times of rabbits inoculated with the virus. This raises ethical concerns about using animals in experiments where death is the end point. We investigated whether or not the opioid analgesic buprenorphine could be used in rabbits without compromising the myxoma virus virulence assay and on the presumption that animals may suffer pain during the course of the disease. Thirty, 5-month-old New Zealand White rabbits were divided into two groups stratified for weight and gender, and inoculated intradermally with 100 pfu of the Standard Laboratory Strain (SLS) of myxoma virus. At day 6 post infection (p.i.), when eyelid swelling was first seen, each animal in one group was treated with 0.03 mg/kg buprenorphine, subcutaneously, morning and evening until death. Animals in the other group were untreated. Animals were weighed daily and rectal temperatures taken morning and evening. Intake of food and water was assessed as was general demeanor including respiratory effort. There was no significant difference in mean survival time, weight change, or demeanor between the two groups. Increased respiratory effort was seen from day 10 p.i. in animals surviving up to and beyond that time but again there was no difference between groups. Animals treated with buprenorphine refused food and water a day earlier than untreated animals, and had lower temperatures immediately prior to death. It was concluded that the opiate analgesic buprenorphine can be used without compromising the current virulence assay for the SLS of myxoma virus in New Zealand White rabbits but that the clinical signs of myxomatosis that could be attributed to pain were not abrogated.     

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.     

A virulence factor of myxoma virus colocalizes with NF-kappaB in the nucleus and interferes with inflammation

NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.     

Myxoma virus expressing human interleukin-12 does not induce myxomatosis in European rabbits

Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressing the complete repertoire of MV proteins. Thus, vMyxIL-12 exhibits promise as an oncolytic candidate and is safe in all known vertebrate hosts, including lagomorphs.