Naturally occurring substitutions in the P/V gene convert the noncytopathic paramyxovirus simian virus 5 into a virus that induces alpha/beta interferon synthesis and cell death

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.     

Simian virus 5 is a poor inducer of chemokine secretion from human lung epithelial cells: identification of viral mutants that activate interleukin-8 secretion by distinct mechanisms

We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI-) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.     

Role for the phosphoprotein P subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses

Six amino acid substitutions in the shared N-terminal region of the P subunit of the viral polymerase and the accessory V protein convert the noncytopathic paramyxovirus simian virus 5 (SV5), which is a poor inducer of host cell responses, into a P/V mutant (P/V-CPI-) that induces high levels of apoptosis, interferon-beta (IFN-beta), and proinflammatory cytokines. In this study, we addressed the question of whether these new mutant phenotypes are due to the presence of an altered P protein or of an altered V protein or of both proteins. By the use of the P/V-CPI- mutant as a backbone, new mutant viruses were engineered to express the wild-type (WT) V protein (+V-wt) or WT P protein (+P-wt) from an additional gene inserted between the HN and L genes. In human epithelial cell lines, the +V-wt virus showed reduced activation of apoptosis and lower secretion of IFN-beta and proinflammatory cytokines compared to the parental P/V-CPI- virus. The presence of a V protein lacking the C-terminal cysteine-rich domain (corresponding to the SV5 I protein) did not reduce these host cell responses to P/V-CPI- infection. Unexpectedly, the +P-wt virus, which expressed a WT P subunit of the viral polymerase, also induced much lower levels of host cell responses than the parental P/V-CPI- mutant. For both +V-wt and +P-wt viruses, reduced levels of IFN-beta synthesis correlated with reduced IRF-3 dimerization and nuclear localization of IRF-3 and NF-kappaB, suggesting that the WT P and V proteins acted at an early stage in antiviral pathways. Host cell responses induced by the various P/V mutants directly correlated with levels of viral mRNA accumulation but not with steady-state levels of genomic RNA. Our results support the hypothesis that WT P and V proteins limit induction of antiviral responses by controlling the production of key viral inducers. A model is presented for the mechanism by which both the P subunit of the viral polymerase and the V accessory protein contribute to the ability of a paramyxovirus to limit activation of antiviral responses.     

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8⁺ and CD4⁺ allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.     

The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.     

Conserved cysteine-rich domain of paramyxovirus simian virus 5 V protein plays an important role in blocking apoptosis

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VDeltaC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VDeltaC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VDeltaC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VDeltaC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VDeltaC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VDeltaC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VDeltaC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VDeltaC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VDeltaC can trigger cell death by inducing ER stress.     

Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of na?ve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity.     

Paramyxovirus-induced shutoff of host and viral protein synthesis: role of the P and V proteins in limiting PKR activation

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI⁻ mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI⁻ mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI⁻ virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI⁻ mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI⁻ mutant was restored to ~50% that of control HeLa cells. By contrast, the rates of P/V-CPI⁻ viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI⁻ mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI⁻ mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.     

A hyperfusogenic F protein enhances the oncolytic potency of a paramyxovirus simian virus 5 P/V mutant without compromising sensitivity to type I interferon

Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI⁻) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI⁻ as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI⁻-G3A) was more fusogenic than the parental P/V-CPI⁻ mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI⁻-G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI⁻ mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI⁻-G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI⁻ mutant and the hyperfusogenic P/V-CPI⁻-G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI⁻-G3A mutant was more effective than P/V-CPI⁻ at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI⁻ were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Polarization of allogeneic T-cell responses by influenza virus-infected dendritic cells

The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-gamma) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-gamma levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.     

IRF family proteins and type I interferon induction in dendritic cells

Dendritic cells （DC）, although a minor population in hematopoietic cells, produce type I interferons （IFN） and other cytokines and are essential for innate immunity. They are also potent antigen presenters and regulate adaptive immunity. Among DC subtypes plasmacytoid DC （pDC） produce the highest amounts of type I IFN. In addition, pro- and anti-inflammatory cytokines such as IL-12 and IL-10 are induced in DC in response to Toll like receptor （TLR） signaling and upon viral infection. Proteins in the IRF family control many aspects of DC activity. IRF-8 and IRF-4 are essential for DC development. They differentially control the development of four DC subsets. IRF-8^-/- mice are largely devoid of pDC and CD8α^＋ DC, while IRF-4^-/- mice lack CD4^＋ DC. IRF-8^-/-, IRF4^-/-, double knock-out mice have only few CD8α CD4^-DC that lack MHC Ⅱ. IRF proteins also control type Ⅰ IFN induction in DC. IRF-7, activated upon TLR signaling is required for IFN induction not only in pDC, but also in conventional DC （cDC） and non-DC cell types. IRF-3, although contributes to IFN induction in fibroblasts, is dispensable in IFN induction in DC. Our recent evidence reveals that type Ⅰ IFN induction in DC is critically dependent on IRF-8, which acts in the feedback phase of IFN gene induction in DC. Type Ⅰ IFN induction in pDC is mediated by MyD88 dependent signaling pathway, and differs from pathways employed in other cells, which mostly rely on TLR3 and RIG-Ⅰ family proteins. Other pro-inflammatory cytokines are produced in an IRF-5 dependent manner. However, IRF-5 is not required for IFN induction, suggesting the presence of separate mechanisms for induction of type Ⅰ IFN and other pro-inflammatory cytokines. IFN and other cytokines produced by activated DC in turn advance DC maturation and change the phenotype and function of DC. These processes are also likely to be governed by IRF family proteins.     

A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.     

Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.     

Matrix protein mutant of vesicular stomatitis virus stimulates maturation of myeloid dendritic cells

Matrix (M) protein mutants of vesicular stomatitis virus have recently been used as oncolytic viruses for tumor therapies and are being developed as vaccine vectors for heterologous antigens. Because dendritic cell (DC) maturation is an important correlate of tumor immunosurveillance and vaccine efficacy, we sought to determine the ability of a recombinant M protein mutant virus (rM51R-M virus) to mature DC in vitro. We have previously shown that rM51R-M virus is defective at inhibiting host gene expression in several cell lines compared to its recombinant wild-type counterpart, rwt virus. Therefore, rM51R-M virus allows the expression of genes involved in antiviral responses, such as the type I interferon (IFN) gene. Our results demonstrate that, in contrast to the rwt virus, rM51R-M virus induced the maturation of myeloid DC (mDC) populations, as indicated by an increase in the surface expression of CD40, CD80, and CD86 as well as the secretion of interleukin-12 (IL-12), IL-6, and type I IFN. In addition, mDC infected with rM51R-M virus effectively activated na?ve T cells in vitro, whereas rwt virus-infected mDC were defective in antigen presentation. The inability of rwt virus to induce mDC maturation was correlated with the inhibition of host gene expression in rwt virus-infected cells. Our studies also indicated that the production of costimulatory molecules on mDC by rM51R-M virus was dependent on the type I IFN receptor, while maturation induced by this virus was largely independent of MyD88. These data indicate that rM51R-M virus effectively stimulates the maturation of mDC and has the potential to promote effective T-cell responses to vector-expressed antigens, activate DC at tumor sites during therapy, and aid in tumor immunosurveillance and destruction.