Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells

Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.     

The Myxoma Virus M-T5 Ankyrin Repeat Host Range Protein Is a Novel Adaptor That Coordinately Links the Cellular Signaling Pathways Mediated by Akt and Skp1 in Virus-Infected Cells

Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.Myxoma virus (MYXV) is a rabbit-specific poxvirus that has proven to be a useful model system to study the mechanism by which virus-encoded immunoregulatory proteins function to manipulate the various host immune responses during the course of viral infection (50). In its long-term evolutionary host (Sylvilagus sp.), MYXV causes a benign disease localized to the site of inoculation, but when the virus infects European rabbits (Oryctolagus cuniculus), it causes a rapid systemic and highly lethal infection called myxomatosis (13). The success of MYXV as a pathogen can be attributed to the ability of the virus to effectively avoid recognition and clearance by the immune systems of susceptible rabbit hosts. At the level of individual virus-infected cells, poxviruses, like MYXV, are particularly adept at binding and entering most mammalian cells, where they attempt to establish a favorable intracellular environment, which promotes viral replication. Thus, the ability of poxviruses to reconfigure or disable the various host antiviral responses of the infected cell directly dictates the outcome of a viral infection at the cellular level (28). To this end, poxviruses possess a large genomic capacity, and all encode a unique repertoire of immunoregulatory and host-interactive proteins that have evolved to specifically mediate a broad range of cellular processes critical for successful viral replication. To date, a large collection of poxvirus-encoded immunoregulatory proteins have been identified and characterized, including virokines, viroreceptors, signaling modulators, and inhibitors of various antiviral responses, such as apoptotic pathways and interferon signaling (43). More recently, a novel category of poxvirus ankyrin (ANK) repeat proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection (48, 49, 53).With the exception of poxviruses, the ANK motif is not commonly reported in viruses, although numerous examples have been identified in eukaryotic, bacterial, and archaeal proteins (6). The ANK motif, a tandemly repeated consensus module of approximately 33 amino acid residues, has been demonstrated to mediate diverse protein-protein interactions between cellular proteins having a broad spectrum of functional roles (32, 42). Solved crystal structures have revealed a conserved fold structure of the ANK repeat unit, by which each repeat forms a characteristic helix-loop-helix structure with a beta-hairpin/loop region projecting out from the helices at a 90° angle (3, 16, 19, 26). However, the ANK fold appears to be defined by its structure rather than any conserved biological function since there is no specific conserved substrate or binding partner structure that is universally recognized by members of the superfamily.The majority of poxviral ANK repeat-containing proteins also include a conserved carboxy-terminal PRANC (pox protein repeats of ankyrin C terminus) motif, which closely resembles a cellular protein motif called the F-box domain (30). Characterized as substrate adaptors, F-box-containing host proteins function to recruit cellular substrate proteins to the SCF ubiquitin-ligase complex (named after their main components, Skp1, cullin 1 [CUL1], and an F-box protein), where the substrates selected by the complex are ubiquitinated and targeted for degradation by the proteasome (21, 45, 60). The process of selective ubiquitination is an essential regulatory step for many cellular processes, and the human genome encodes more than 70 different F-box proteins, which collectively are thought to specifically target a broad collection of cellular substrates for delivery to the SCF complex to initiate turnover (62).Accounting for the largest family of poxviral proteins, almost all chordopoxviruses encode multiple ANK repeat-containing proteins, some of which have been defined as viral host range or virulence factors (30). For example, canarypox virus encodes 51 ANK repeat proteins, accounting for greater than 20% of the genome; however, most other poxviruses express less than a half dozen ANK repeat proteins (52). MYXV encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which have been described as virulence factors for myxomatosis in rabbits (5, 8, 33). The MYXV host range factor M-T5 was first characterized for its ability to regulate viral tropism within rabbit lymphocytes and, later, some classes of human cancer cell lines (33, 51). In human cancer cells, the direct physical interaction between M-T5 and the host cell Akt was shown to be a key restriction determinant for MYXV tropism in a subset referred to as type II cancer cells (56). Furthermore, M-T5 was shown to be functionally interchangeable with a host ANK repeat protein called PIKE-A, and the activation of Akt by either the viral M-T5 or the host PIKE-A protein was critical for MYXV permissiveness in type II human cancer cells (57). M-T5 was also demonstrated to protect MYXV-infected cells from virus-induced cell cycle arrest, a property which was linked to its ability to interact with a member of the host cell SCF complex called CUL1 (20). Unlike M-T5, no specific host binding partners or target substrates have yet been identified for M148, M149, or M150. However, in tumor necrosis factor alpha (TNF-α)-stimulated cells, M150 was shown to colocalize in the nucleus with NF-κB p65, suggesting that this MYXV protein may modulate the NF-κB pathway (8).In this study, we demonstrate that M-T5, M148, M149, and M150 all have functional carboxy-terminal PRANC/F-box-like domains and that each one can interact directly with the Skp1 component of the host SCF complex. We further examined the various binding domains of M-T5 and identified ANK repeats I and II as being important for interaction with Akt, whereas the PRANC/F-box-like domain was essential for binding to Skp1. We also show that the previously reported interaction of M-T5 with CUL1 was in fact, indirect linking of M-T5 to the host SCF complex via Skp1. More specifically, we investigated the ability of M-T5 to function as a molecular scaffold to link disparate cellular binding partners together within a single complex and report that the viral protein binds Akt and the SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we demonstrate that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during MYXV infection. These results suggest that ANK/PRANC proteins, such as M-T5, play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for viral replication.     

M-T5, the ankyrin repeat, host range protein of myxoma virus, activates Akt and can be functionally replaced by cellular PIKE-A

The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA 103:4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.     

Pharmacological Manipulation of the Akt Signaling Pathway Regulates Myxoma Virus Replication and Tropism in Human Cancer Cells

Viruses have evolved an assortment of mechanisms for regulating the Akt signaling pathway to establish a cellular environment more favorable for viral replication. Myxoma virus (MYXV) is a rabbit-specific poxvirus that encodes many immunomodulatory factors, including an ankyrin repeat-containing host range protein termed M-T5 that functions to regulate tropism of MYXV for rabbit lymphocytes and certain human cancer cells. MYXV permissiveness in these human cancer cells is dependent upon the direct interaction between M-T5 and Akt, which has been shown to induce the kinase activity of Akt. In this study, an array of compounds that selectively manipulate Akt signaling was screened and we show that only a subset of Akt inhibitors significantly decreased the ability of MYXV to replicate in previously permissive human cancer cells. Furthermore, reduced viral replication efficiency was correlated with lower levels of phosphorylated Akt. In contrast, the PP2A-specific phosphatase inhibitor okadaic acid promoted increased Akt kinase activation and rescued MYXV replication in human cancer cells that did not previously support viral replication. Finally, phosphorylation of Akt at residue Thr308 was shown to dictate the physical interaction between Akt and M-T5, which then leads to phosphorylation of Ser473 and permits productive MYXV replication in these human cancer cells. The results of this study further characterize the mechanism by which M-T5 exploits the Akt signaling cascade and affirms this interaction as a major tropism determinant that regulates the replication efficiency of MYXV in human cancer cells.Following viral infection, substantial alterations in cellular physiology often lead to modification of various cellular pathways critical to the success of viral replication. The demands for energy, nutrients, and macromolecular synthesis that accompany viral replication can be substantial; thus, many viruses have evolved elaborate strategies for hijacking key cellular signaling networks necessary to support their demands (9). By the same token, antiviral pathways activated by the virus infection may also need to be blocked or subverted to ensure successful virus replication. Poxviruses possess large double-stranded DNA (dsDNA) genomes that encode multiple gene products that specifically modify or debilitate the various host signaling responses of the infected cell (28). Many of the immunoregulatory factors expressed by poxviruses have been well characterized, and these factors include virokines, viroreceptors, signaling modulators, and inhibitors of various antiviral responses, such as initiation of apoptosis pathways and signaling by protective cytokines, like interferon and tumor necrosis factor (TNF) (42).Myxoma virus (MYXV) is a member of the Leporipoxvirus genus and exhibits a restricted pathogenesis that is limited to rabbits, primarily due to its specific immunomodulation of the immune system of leporids (48). In rabbits (Sylvilagus spp.) of the Americas, MYXV infection results in a benign infection, characterized by a cutaneous fibroma restricted to the site of inoculation (14); however, the same virus causes a rapid systemic and highly lethal infection called myxomatosis in European rabbits (Oryctolagus cuniculus) (15). Although MYXV has a narrow host range in nature and is pathogenic only to European rabbits, the tropism of MYXV has recently been extended to include human tumor cells in vitro (6, 47, 54, 57, 60) and in xenografted mice in vivo (24, 25, 61). The mechanisms that mediate MYXV tropism in human cancer cells are still being investigated, but one signaling requirement has been linked to the state of cellular Akt kinase activity (57). Human cancer cells (called type I) that exhibit high levels of endogenous phosphorylated Akt (Ser473 and Thr308) supported permissive MYXV replication, while cells with no detectable endogenous phosphorylated Akt, which were unaffected by the virus infection, were nonpermissive (type III). A unique subset of cancer cells (type II) were found to be permissive to wild-type MYXV but did not support MYXV replication following the deletion of the viral host range factor M-T5 (vMyxT5KO). These type II cells constitutively expressed only low levels of endogenous phosphorylated Akt (mostly at Thr308), but following infection with permissive MYXV, a significant increase in Akt phosphorylation (particularly at Ser473) was observed. In stark contrast, the endogenous levels of phosphorylated Akt remained essentially unchanged when type II cells were infected with the nonpermissive M-T5 knockout virus MYXV (vMyxT5KO) (57).The host range factor M-T5 is essential for MYXV replication in rabbit primary lymphocytes (RL-5 cells) and for virus pathogenesis in European rabbits (31). Structurally, M-T5 possesses seven ankyrin (ANK) repeats and a carboxyl-terminal PRANC (pox protein repeats of ankyrin C-terminal) motif, which closely resembles a cellular protein motif called the F-box domain (29). Interaction between M-T5 and components of the cellular SCF (Skp-cullin-F-box) ubiquitin ligase complex was shown to protect MYXV-infected cells from cell cycle arrest (19). In MYXV-infected type II human cancer cells, physical interaction between M-T5 and cellular Akt was shown to upregulate the kinase activity of Akt (57). In another study, M-T5 was shown to be functionally interchangeable with the host ANK repeat-containing protein PIKE-A, and activation of Akt by either PIKE-A or the viral M-T5 protein was sufficient to mediate MYXV permissiveness in type II human cancer cells (59). Similarly, addition of the immunosuppressant drug rapamycin was successful at rescuing vMyxT5KO replication in type II cells by upregulating Akt activation through the mTOR pathway (47). The critical role of Akt in the regulation of multiple biological processes makes Akt a central regulator of cellular signaling, and therefore, it is not surprising that many viruses have developed sophisticated strategies for manipulating the activation of Akt (9, 11).The serine/threonine kinase Akt (also called protein kinase B [PKB]) was initially discovered as the cellular homolog of the viral oncogene (v-Akt) carried by the AKT8 retrovirus isolated from a murine T-cell lymphoma (7, 20, 46). There are three isoforms found in mammals (Akt1, -2, and -3), encoded by separate genes but sharing over 80% amino acid sequence identity. Activation of Akt is predominantly dependent upon phosphoinositide 3-kinase (PI3K), which phosphorylates phosphoinositides (PIs) at the D3 position of the inositol ring to generate PI(3,4,5)P₃ (PIP₃). Akt possesses an N-terminal PH (pleckstrin homology) domain that binds PIP₃ to promote its translocation of the plasma membrane. Once localized at the membrane, Akt becomes phosphorylated at residue Thr308 in the activation loop by phosphoinositide-dependent kinase 1 (PDK1) and also within the carboxy terminus at residue Ser473 by mTORC2 (mammalian target of rapamycin complex 2) (2, 49, 50). Phosphorylation of both sites is necessary for full induction of Akt kinase activity. Akt is a key regulator of many important cellular functions, including cell survival, proliferation, glucose metabolism, and protein synthesis. In the majority of human cancer cells, the Akt pathway is either mutated or constitutively activated, contributing to cancer progression through both stimulation of cellular proliferation and inhibition of apoptosis (34, 55).In this study, we screened an array of Akt inhibitor compounds that selectively manipulate the Akt signaling network at some level and report that certain Akt inhibitors significantly blocked MYXV replication in previously permissive type I and II human cancer cells. An additional set of inhibitors selectively inhibited only the replication of MYXV deleted for M-T5 and did not modify the replicative ability of the parental wild-type virus. Furthermore, the decrease in viral replication efficiency was correlated with lower levels of phosphorylated Akt at residues Thr308 and Ser473. In contrast, certain PP2A-specific phosphatase inhibitors, such as okadaic acid, promoted increased Akt kinase activation and rescued MYXV replication in type III human cancer cells that did not previously support viral replication. Finally, we demonstrate that the hemi-phosphorylation of Akt at residue Thr308 dictates physical interaction between Akt and M-T5, which ultimately leads to productive MYXV replication in type II cancer cells. These studies show that activation of the Akt signaling cascade is essential for efficient MYXV replication in human cancer cells and further demonstrate the dynamic role by which M-T5 manipulates Akt signaling to establish a cellular environment more favorable for viral replication.     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

The host phosphoinositide 5-phosphatase SHIP2 regulates dissemination of vaccinia virus

After fusing with the plasma membrane, enveloped poxvirus virions form actin-filled membranous protrusions, called tails, beneath themselves and move toward adjacent uninfected cells. While much is known about the host and viral proteins that mediate formation of actin tails, much less is known about the factors controlling release. We found that the phosphoinositide 5-phosphatase SHIP2 localizes to actin tails. Localization requires phosphotyrosine, Abl and Src family tyrosine kinases, and neural Wiskott-Aldrich syndrome protein (N-WASP) but not the Arp2/Arp3 complex or actin. Cells lacking SHIP2 have normal actin tails but release more virus. Moreover, cells infected with viral strains with mutations in the release inhibitor A34 release more virus but recruit less SHIP2 to tails. Thus, the inhibitory effects of A34 on virus release are mediated by SHIP2. Together, these data suggest that SHIP2 and A34 may act as gatekeepers to regulate dissemination of poxviruses when environmental conditions are conducive.     

Role of the serine-threonine kinase PAK-1 in myxoma virus replication

Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry. In contrast, late virus gene expression and viral DNA replication were selectively compromised in restrictive 3T3 cells. Addition of specific protein kinase inhibitors, many of which shared the ability to influence the activity of the STKs p21-activated kinase 1 (PAK-1) and Raf-1 attenuated MV replication in permissive 3T3 cells. Western blot detection of the phosphorylated forms of PAK-1 (Thr423) and Raf-1 (Ser338) confirmed activation of these kinases in permissive cells after MV infection or gamma interferon treatment, but the activated forms of both kinases were greatly reduced or absent in restrictive 3T3 cells. The biological significance of these activations was demonstrated by using the autoinhibitory domain of PAK-1 (amino acids 83 to 149), expression of which reduced the efficiency of MV infection in permissive 3T3 cells concurrent with a decrease in PAK-1 activation. In comparison, overexpression of a constitutively active PAK-1 (T423E) mutant increased MV replication in restrictive 3T3 cells. These observations suggest that induced signaling via cellular STKs may play important roles in determining the permissiveness of host cells to poxvirus infection.     

痘病毒介导的NF-κB信号通路的调控

痘病毒是人和多种动物痘病的病原体,作为嗜上皮型的DNA病毒,在进化过程中,痘病毒特定的基因产物作用于机体免疫调控系统,调节免疫反应的进程和作用方式,进而完成其感染细胞、复制和繁殖的生物学过程。NF-κB信号通路是机体免疫系统重要的信号转导调节系统,各种痘病毒采用自身特殊的策略作用于这一免疫调节的重要靶系统,应对机体对其免疫清除和免疫反应。对痘病毒介导的宿主免疫调节的深入研究有助于研发新型疫苗和治疗性制剂。本文对近十年来各种痘病毒编码的多种目标蛋白参与宿主NF-κB信号通路调控的分子机制进行综述。     

Poxvirus antagonism of innate immunity by Bcl-2 fold proteins

Poxviruses have evolved numerous mechanisms to evade host innate immunity. Sensory pathways that are activated by Toll-like and nucleotide receptors, as well as innate cell death pathways, are both targets of antagonism by viral proteins. Recent structural, biochemical and functional studies of poxvirus proteins have identified a family of α-helical proteins that adopt a Bcl-2 fold despite highly divergent polypeptide sequences from cellular proteins that regulate apoptosis. These newly identified proteins have assumed new roles in antagonism of NF-κB and interferon signaling pathways and interfere with the release of pro-inflammatory cytokines. Structures of isolated viral proteins and their complexes with cellular targets provide insight into the diverse ways that the Bcl-2 scaffold can be exploited for antagonism of host immunity.     

Myxoma virus M-T5 protects infected cells from the stress of cell cycle arrest through its interaction with host cell cullin-1

The myxoma virus (MV) M-T5 gene encodes an ankyrin repeat protein that is important for virus replication in cells from several species. Insight was gained into the molecular mechanisms underlying the role of M-T5 as a host range determinant when the cell cycle regulatory protein cullin-1 (cul-1) was identified as a cellular binding partner of M-T5 and found to colocalize with the protein in both nuclear and cytosolic compartments. Consistent with this interaction, infection with wild-type MV (vMyxlac) or a deletion mutant lacking M-T5 (vMyxT5KO) differentially altered cell cycle progression in a panel of permissive and nonpermissive cells. Cells infected with vMyxlac transitioned rapidly out of the G0/G1 phase and preferentially accumulated at the G2/M checkpoint, whereas infection with vMyxT5KO impeded progression through the cell cycle, resulting in a greater percentage of cells retained at G0/G1. Levels of the cul-1 substrate, p27/Kip-1, were selectively increased in cells infected with vMyxT5KO compared to vMyxlac, concurrent with decreased phosphorylation of p27/Kip-1 at Thr187 and decreased ubiquitination. Compared to cells infected with vMyxlac, cell death was increased in vMyxT5KO-infected cells following treatment with diverse stimuli known to induce cell cycle arrest, including infection itself, serum deprivation, and exposure to proteasome inhibitors or double-stranded RNA. Moreover, infection with vMyxlac, but not vMyxT5KO, was sufficient to overcome the G0/G1 arrest induced by these stimuli. These findings suggest that M-T5 regulates cell cycle progression at the G0/G1 checkpoint, thereby protecting infected cells from diverse innate host antiviral responses normally triggered by G0/G1 cell cycle arrest.     

Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses.     

Viral subversion of the host protein synthesis machinery

Viruses are fully reliant on the translation machinery of their host cells to produce the polypeptides that are essential for viral replication. Consequently, viruses recruit host ribosomes to translate viral mRNAs, typically using virally encoded functions to seize control of cellular translation factors and the host signalling pathways that regulate their activity. This not only ensures that viral proteins will be produced, but also stifles innate host defences that are aimed at inhibiting the capacity of infected cells for protein synthesis. Remarkably, nearly every step of the translation process can be targeted by virally encoded functions. This Review discusses the diverse strategies that viruses use to subvert host protein synthesis functions and regulate mRNA translation in infected cells.     

Control of Apoptosis by Poxviruses

Poxviruses express a variety of proteins which can function to inhibit apoptosis in infected cells, allowing virus replication to continue and conferring a broad host range. Some poxvirus antiapoptosis proteins act by sequestering or inactivating inducers of apoptosis such as dsRNA and superoxide anions. Others interfere with signaling by receptors including those belonging to the TNF receptor superfamily that would otherwise activate a proteolytic cascade that terminates with the cleavage of death substrates. The cowpox virus crmA protein directly inhibits cysteine proteinases within the cascade and can also block apoptosis triggered by the serine proteinase granzyme B. Finally there are poxvirus antiapoptosis proteins containing ankyrin repeat regions that are thought to interact with cellular proteins.     

Technical knockout: understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes

The study of viral pathogens with genomes as large and complex as poxviruses represents a constant experimental challenge. Advances in recombinant DNA technologies have provided sophisticated methods to produce mutants defective in one or more viral genes, termed knockout (KO) viruses, thereby facilitating research into the impact of specific gene products on viral pathogenesis. Such strategies have rapidly advanced the systematic mining of many poxvirus genomes and enabled researchers to identify and characterize poxvirus genes whose functions represent the culmination of host and pathogen coevolution. Of particular interest are the multiple classes of virus-encoded immunomodulatory proteins that have evolved specifically to allow poxviruses to evade, obstruct or subvert critical elements within the host innate and acquired immune responses. Functional characterization of these viral genes by generating KO viruses and investigating the phenotypic changes that result is an important tool for understanding the molecular mechanisms underlying poxvirus replication and pathogenesis. Moreover, the insights gained have led to new developments in basic and clinical virology, provided a basis for the design of new vaccines and antivirals, and increased the potential application of poxviruses as investigative tools and sources of biotherapeutics for the treatment of human diseases.     

Vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor

The presumed broad tropism of poxviruses has stymied attempts to identify both the cellular receptor(s) and the viral determinant(s) for binding. Detailed studies of poxvirus binding to and infection of primary human cells have not been conducted. In particular, the determinants of target cell infection and the consequences of infection for cells involved in the generation of antiviral immune responses are incompletely understood. In this report, we show that vaccinia virus (VV) exhibits a more restricted tropism for primary hematolymphoid human cells than has been previously recognized. We demonstrate that vaccinia virus preferentially infects antigen-presenting cells (dendritic cells, monocytes/macrophages, and B cells) and activated T cells, but not resting T cells. The infection of activated T cells is permissive, with active viral replication and production of infectious progeny. Susceptibility to infection is determined by restricted expression of a cellular receptor that is induced de novo upon T-cell activation and can be removed from the cell surface by either trypsin or pronase treatment. The VV receptor expressed on activated T cells displays unique characteristics that distinguish it from the receptor used to infect cell lines in culture. The observed restricted tropism of VV may have significant consequences for the understanding of natural poxvirus infection and immunity and for poxvirus-based vaccine development.     

Viral appropriation of apoptotic and NF-kappaB signaling pathways

Viruses utilize a variety of strategies to evade the host immune response and replicate in the cells they infect. The comparatively large genomes of the Orthopoxviruses and gammaherpesviruses encode several immunomodulatory proteins that are homologous to component of the innate immune system of host cells, which are reviewed here. However, the viral mechanisms used to survive host responses are quite distinct between these two virus families. Poxviruses undergo continuous lytic replication in the host cytoplasm while expressing many genes that inhibit innate immune responses. In contrast, herpesviruses persist in a latent state during much of their lifecycle while expressing only a limited number of relatively non-immunogenic viral proteins, thereby avoiding the adaptive immune response. Poxviruses suppress, whereas latent gammaherpesviruses activate, signaling by NF-kappaB, yet both viruses target similar host signaling pathways to suppress the apoptotic response. Here, modulation of apoptotic and NF-kappaB signal transduction pathways are examined as examples of common pathways appropriated in contrasting ways by herpesviruses and poxviruses.     

Modulation of cytokine networks by poxvirus: the myxoma virus model

Poxviruses are large eukaryotic DNA viruses that have evolved a variety of strategies to evade the host immune responses to virus infection. Myxoma virus is of particular interest because it is a rabbit-specific poxvirus that induces a defined systemic virus disease, myxomatosis, in the infected European rabbit. A variety of virus-encoded proteins that are important for subverting cellular immunity and inflammation have been identified, and many of these operate by modulating or subverting cellular cytokine networks. Studies on viral proteins which mediate these immune alterations reveal a multiplicity of strategies by which poxviruses evade clearance from immunocompetent hosts and also identify a new class of immunomodulatory proteins with which to investigate the effector functions of the immune system.     

Poxvirus tumor necrosis factor receptor (TNFR)-like T2 proteins contain a conserved preligand assembly domain that inhibits cellular TNFR1-induced cell death

The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties; secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-alpha, while intracellular M-T2 blocks virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-mediated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-induced death. These cells were also resistant to human TNF-alpha, but M-T2 expression did not alter surface expression levels of TNFRs. Previous studies indicated that T2's antiapoptotic function was conferred by the N-terminal region of the protein, and further examination of this region revealed a highly conserved N-terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-alpha-related apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human TNFRs but does not prevent human TNF-alpha binding to cellular receptors. Thus, M-T2 vPLAD is a species-nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins. Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential target in the development of the next generation of TNF-alpha/TNFR therapeutics.     

How vaccinia virus has evolved to subvert the host immune response

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.     

Host cellular signaling induced by influenza virus

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell’s innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidylinositol- 3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.