R. Mark L. Buller

2′- O methylation of the viral mRNA cap evades host restriction by IFIT family members

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2′-O positions of the 5′ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2′-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2′-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2′-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2′-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2′-O methylation of the 5′ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2′-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

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.

A Poxvirus-Encoded Semaphorin Induces Cytokine Production from Monocytes and Binds to a Novel Cellular Semaphorin Receptor, VESPR

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.

A Poxvirus Protein That Binds to and Inactivates IL-18, and Inhibits NK Cell Response

IL-18 induces IFN-γ and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response.

Human monkeypox: an emerging zoonotic disease

Zoonotic monkeypox virus is maintained in a large number of rodent and, to a lesser extent, nonhuman primate species in West and central Africa. Although monkeypox virus was discovered in 1958, the prototypic human cases were not witnessed until the early 1970s. Before this time, it is assumed that infections were masked by smallpox, which was then widely endemic. Nevertheless, since the 1970s, reported monkeypox virus infections of humans have escalated, as have outbreaks with reported human-to-human transmission. This increase is likely due to numerous factors, such as enhanced surveillance efforts, environmental degradation and human urbanization of areas where monkeypox virus is maintained in its animal reservoir(s) and, consequently, serve as a nidus for human infection. Furthermore, viral genetic predispositions enable monkeypox virus to infect many animal species, represented in expansive geographic ranges. Monkeypox virus was once restricted to specific regions of Africa, but its environ has expanded, in one case intercontinentally--suggesting that human monkeypox infections could continue to intensify. As a zoonotic agent, monkeypox virus is far less sensitive to typical eradication measures since it is maintained in wild-animal populations. Moreover, human vaccination is becoming a less viable option to control poxvirus infections in todays increasingly immunocompromised population, particularly with the emergence of HIV in Sub-Saharan Africa. An increased frequency of human monkeypox virus infections, especially in immunocompromised individuals, may permit monkeypox virus to evolve and maintain itself independently in human populations.

Hexadecyloxypropyl-cidofovir, CMX001, prevents adenovirus-induced mortality in a permissive, immunosuppressed animal model

Adenoviruses (Ads) cause a wide array of end-organ and disseminated diseases in severely immunosuppressed patients. For example, ≈20% of pediatric allogeneic hematopoietic stem cell transplant recipients develop disseminated Ad infection, and the disease proves fatal in as many as 50–80% of these patients. Ad infections are a serious problem for solid-organ transplant recipients and AIDS patients as well. Unfortunately, there are no antiviral drugs approved specifically to treat these infections. A suitable animal model that is permissive for Ad replication would help in the discovery process. Here we identify an animal model to study Ad pathogenesis and the efficacy of antiviral compounds. We show that human serotype 5 Ad (Ad5) causes severe systemic disease in immunosuppressed Syrian hamsters that is similar to that seen in immunocompromised patients. We also demonstrate that hexadecyloxypropyl-cidofovir (CMX001) rescues the hamsters from a lethal challenge with Ad5. The antiviral drug provided protection both prophylactically and when given up to 2 days after i.v. exposure to Ad5. CMX001 acts by reducing Ad replication in key target organs. Thus, the immunosuppressed Syrian hamster is a powerful model to evaluate anti-Ad drugs, and its use can facilitate the entry of drugs such as CMX001 into clinical trials.

Cell proliferative response to vaccinia virus is mediated by VGF.

VGF, a polypeptide encoded by vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors EGF and TGF-alpha. The availability of a VGF minus (VGF-) virus mutant has enabled us to examine the role of VGF in the replication of virus in vitro and in vivo. Studies in vitro with A431 cells (high EGF receptor density) showed that VGF+ wild-type virus induced the rapid formation of a focus of infection (not a plaque) which could be blocked by a monoclonal antibody to the EGF receptor. In vivo experiments with chicken embryos indicated that VGF+ virus stimulated the growth of ectodermal and entodermal cells of the chorioallantoic membrane. At early times, the majority of proliferating cells contained no detectable virus antigen, indicating that cell growth preceded infection and was a consequence of VGF secretion. Relative to VGF- virus, VGF+ virus produced lesions which contained more proliferating cells, more virus antigen, and increased amounts of infectious progeny. Secretion of VGF thus explains the conundrum of a nontransforming, strongly cytopathic virus inducing a hyperplastic cell response.

Induction of Natural Killer Cell Responses by Ectromelia Virus Controls Infection

ABSTRACT Natural killer (NK) cells play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses. In poxvirus infections, the role of NK cells is less clear. We examined disease progression in C57BL/6 mice after the removal of NK cells by both antibody depletion and genetic means. We found that NK cells were crucial for survival and the early control of virus replication in spleen and to a lesser extent in liver in C57BL/6 mice. Studies of various knockout mice suggested that γδ T cells and NKT cells are not important in the C57BL/6 mousepox model and CD4+ and CD8+ T cells do not exhibit antiviral activity at 6 days postinfection, when the absence of NK cells has a profound effect on virus titers in spleen and liver. NK cell cytotoxicity and/or gamma interferon (IFN-γ) secretion likely mediated the antiviral effect needed to control virus infectivity in target organs. Studies of the effects of ectromelia virus (ECTV) infection on NK cells demonstrated that NK cells proliferate within target tissues (spleen and liver) and become activated following a low-dose footpad infection, although the mechanism of activation appears distinct from the ligand-dependent activation observed with MCMV. NK cell IFN-γ secretion was detected by intracellular cytokine staining transiently at 32 to 72 h postinfection in the lymph node, suggesting a role in establishing a Th1 response. These results confirm a crucial role for NK cells in controlling an ECTV infection.

The genomic sequence of ectromelia virus, the causative agent of mousepox.

Ectromelia virus is the causative agent of mousepox, an acute exanthematous disease of mouse colonies in Europe, Japan, China, and the U.S. The Moscow, Hampstead, and NIH79 strains are the most thoroughly studied with the Moscow strain being the most infectious and virulent for the mouse. In the late 1940s mousepox was proposed as a model for the study of the pathogenesis of smallpox and generalized vaccinia in humans. Studies in the last five decades from a succession of investigators have resulted in a detailed description of the virologic and pathologic disease course in genetically susceptible and resistant inbred and out-bred mice. We report the DNA sequence of the left-hand end, the predicted right-hand terminal repeat, and central regions of the genome of the Moscow strain of ectromelia virus (approximately 177,500 bp), which together with the previously sequenced right-hand end, yields a genome of 209,771 bp. We identified 175 potential genes specifying proteins of between 53 and 1924 amino acids, and 29 regions containing sequences related to genes predicted in other poxviruses, but unlikely to encode for functional proteins in ectromelia virus. The translated protein sequences were compared with the protein database for structure/function relationships, and these analyses were used to investigate poxvirus evolution and to attempt to explain at the cellular and molecular level the well-characterized features of the ectromelia virus natural life cycle.

Inhibition of an inflammatory response is mediated by a 38-kDa protein of cowpox virus

The Brighton Red (BR) strain of cowpox virus induces a flat, bright red pock on the chorioallantoic membrane (CAM) of the 12-day-old chick embryo. In contrast, mutants with a deleted 38K gene (which is located 31 to 32 kb from the right-hand end of the virus genome) induced a raised, white, and opaque pock. During the first 24-hr p.i., both CPV-BR and the 38K deletion mutants replicated similarly in the CAM of the chick embryo, as indicated by immunocytochemical detection of similar amounts of virus antigen. By 48 hr p.i., the pocks induced by the mutant and CPV-BR are strikingly different. The pocks induced by the 38K deletion mutants were infiltrated by large numbers of heterophils and macrophages, which correlated with a reduction in the levels of virus antigen and virus infectivity. The CPV-BR pock had an absence of inflammatory cells and increased levels of virus antigen and infectivity. By 72 hr p.i., many of the pocks induced by the mutant were undergoing resolution of the virus infection, as indicated by further decrease of virus antigen and visible signs of healing, whereas CPV-BR pocks continued to be a site of active viral replication. These data are consistent with a model where this 38-kDa protein directly or indirectly inhibits the generation of chemotactic molecules which are elicited during virus replication in the CAM or, alternatively, blocks the interaction of these molecules with cells of the host inflammatory response.