PKR protection against intranasal vesicular stomatitis virus infection is mouse strain dependent.

The interferon-induced antiviral state is mediated by interferon-stimulated genes that are upregulated in concert after stimulation by type I interferons. Because so many viruses encode strategies to inactivate the interferon-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR, this protein is likely to be a major player in antiviral defense. Here we demonstrate the increased susceptibility of PKR-/- animals to vesicular stomatitis virus (VSV) by the intranasal route, but also demonstrate that the protective effects of PKR are mouse strain dependent. We have found the difference between wild-type-BALB/c and 129SvEv animals to be on the order of 5 logs, with high levels of virus present in the lungs of BALB/c but not 129SvEv animals. To evaluate the sensitivity of PKR-/- mice to VSV clearly, the PKR mutation was bred onto the resistant 129SvEv background. The increased sensitivity of PKR-/- mice, compared to PKR+/+ strain-matched controls, is on the order of 10-fold as measured by median lethal dose (LD50). PKR-/- 129 mice support VSV replication in the lung unlike controls. While this result clearly demonstrates an important role for PKR in protection against VSV infection of the lung, it also underlines the importance of other host factors in containing a viral infection.

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.

Enhanced antiviral and antiproliferative properties of a STAT1 mutant unable to interact with the protein kinase PKR.

We have previously reported a physical association between STAT1 and the protein kinase double-stranded RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a manner independent of PKR kinase activity. In this report, we have further characterized the properties of both molecules by mapping the sites of their interaction. A STAT1 mutant unable to interact with PKR displays enhanced interferon gamma (IFN-gamma)-induced transactivation capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1(-/-) cells enhances both the antiviral and antiproliferative effects of IFNs as opposed to STAT1. We also provide evidence that STAT1 functions as an inhibitor of PKR in vitro and in vivo. That is, phosphorylation of eIF-2alpha is enhanced in STAT1(-/-) than STAT1(+/+) cells in vivo, and this correlates with higher activation capacity of PKR in STAT1(-/-) cells. Genetic experiments in yeast demonstrate the inhibition of PKR activation and eIF-2alpha phosphorylation by STAT1 but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in translational control through the modulation of PKR activation and eIF-2alpha phosphorylation.

Control of Sindbis virus infection by antibody in interferon-deficient mice.

Antibodies clear Sindbis virus from infected animals through an unknown mechanism. To determine whether interferon-induced pathways are required for this clearance, we examined mice which are unable to respond to alpha/beta interferon or gamma interferon. Although extremely susceptible to infection, such mice survived and completely cleared virus if antibodies against Sindbis virus were given.

Type I IFN modulates innate and specific antiviral immunity.

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

Interferon alpha/beta-mediated inhibition and promotion of interferon gamma: STAT1 resolves a paradox.

Induction of high systemic levels of type 1 interferons (IFNs) IFN-alpha and IFN-beta is a hallmark of many viral infections. In addition to their potent antiviral effects, these cytokines mediate a number of immunoregulatory functions and can promote IFN-gamma expression in T cells. However, during viral infections of mice IFN-gamma production is not always observed at the same time as systemic IFN-alpha/beta production and when, elicited at these times, is IFN-alpha/beta-independent. We demonstrate that type 1 interferons not only fail to induce, but also act to inhibit, IFN-gamma expression by both NK and T cells. The mechanism of inhibition is dependent upon the IFN-alpha/beta receptor and the signal transducer and activator of transcription 1 (STAT1). In the absence of STAT1, not only are the IFN-alpha/beta-mediated inhibitory effects completely abrogated, but the cytokines themselves can induce IFN-gamma expression. These results indicate that endogenous biochemical pathways are in place to negatively regulate NK and T cell IFN-gamma expression elicited by IFN-alpha/beta or other stimuli, at times of innate responses to viral infections. They also show that type 1 interferon signaling can occur through STAT1-dependent and independent mechanisms and suggest that efficient induction of IFN-gamma expression by IFN-alpha/beta requires STAT1 regulation. Such immunoregulatory pathways may be critical for shaping the endogenous innate and virus-specific adaptive immune responses to viral infections.

Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7.

Interferon (IFN) genes are among the earliest transcriptional responses to virus infection of mammalian cells. Although the regulation of the IFNbeta gene has been well characterized, the induction of the large family of IFNalpha genes has remained obscure. We report that the IFNalpha genes can be divided into two groups: an immediate-early response gene (IFNalpha4) which is induced rapidly and without the need for ongoing protein synthesis; and a set of genes that display delayed induction, consisting of at least IFNalpha2, 5, 6 and 8, which are induced more slowly and require cellular protein synthesis. One protein that must be synthesized for induction of the delayed gene set is IFN itself, presumably IFNalpha4 or IFNbeta, which stimulates the Jak-Stat pathway through the IFN receptor, resulting in activation of the transcription factor interferon-stimulated gene factor 3 (ISGF3). Among the IFN-stimulated genes induced through this positive feedback loop is the IFN regulatory factor (IRF) protein, IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus-specific signals, involving two C-terminal serine residues, are required for induction of the delayed IFNalpha gene set.

The role of interferon in influenza virus tissue tropism.

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.

Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems.

The NS1 protein is the only nonstructural protein encoded by influenza A virus. It has been proposed that the NS1 performs several regulatory functions during the viral replication cycle, including the regulation of synthesis, transport, splicing, and translation of mRNAs. Through the use of reverse genetics, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated. Our results indicate that the NS1 of influenza A virus is an auxiliary (virulence) factor which plays a crucial role in inhibiting interferon-mediated antiviral responses of the host.

ISGF3 gamma p48, a specificity switch for interferon activated transcription factors.

Interferon (IFN) induces gene expression by phosphorylating latent transcription factors of the STAT family. Two different STAT multimeric complexes that bind distinct enhancer elements are activated by IFN alpha and IFN gamma, dictated by the DNA-binding protein ISGF3 gamma p48. This protein, a member of the IFN regulatory factor (IFR) family, acts as an adaptor protein to redirect STAT multimers from their intrinsic palindromic sequence specificity to interactions with a composite element composed of an IRF site juxtaposed with a STAT half-site. Sequence similarity within the IRF family suggests that other members could serve as adaptor proteins for transcriptional activators. Recent evidence suggests that PIP (LSIRF) sequesters the Ets protein PU.1 at a composite DNA element lends support to this adaptor hypothesis.

Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease.

The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens.

Cytoplasmic transcription factors: mediators of cytokine signaling.

The distinct pattern of transcriptional responses of cells to different extracellular signals requires a signal transduction pathway that provides rapid, accurate, and faithful transmission of information from the cell surface to the nucleus. One mechanism exploited by many cytokines, exemplified by interferons (IFN) but also used by many interleukins and growth factors, uses a family of cytoplasmic transcription factors that are activated by tyrosine phosphorylation. Once phosphorylated by receptor-associated tyrosine kinases, these proteins assemble into multimeric transcription factors, translocate to the nucleus, and bind specific DNA sequence elements in the promoters of target genes.

Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors.

Cytokines and growth factors elicit responses in target cells through induction of gene expression. Signaling mechanisms leading to gene transcription from cell surface receptors often require tyrosine phosphorylation. A family of transcription factors comprising the interferon (IFN)-stimulated gene factor 3 (ISGF3) multimeric complex are phosphorylated and activated in response to interferon. We describe a protein 50% identical to the 91-kDa subunit of ISGF3 that constitutes the acute phase response factor (APRF). This protein was rapidly activated by interleukin-6 to bind an enhancer element common to genes activated in liver cells during the acute phase response to inflammation. Remarkably, APRF was also activated by IFN alpha, IFN gamma, epidermal growth factor, platelet-derived growth factor, colony stimulating factor-1, and the cytokines leukemia inhibitory factor and oncostatin M. The growth factors also activated a third, distinct but related, DNA-binding protein in addition to APRF and p91. This novel factor or a closely related one, but neither APRF nor p91, was also activated in lymphoid cells by interleukin-2, erythropoietin, and interleukin-3. Activation of APRF, p91, and additional members of the ISGF3 family is thus a general feature of a wide variety of signaling pathways, integrating diverse signals through common transcriptional regulators.

Identification of cDNAs corresponding to mosquito ribosomal protein genes.

Sequences encoding mosquito (Aedes albopictus) ribosomal proteins L8, L14 and L31 were identified from a cDNA library made from size-selected polyadenylated mRNA. Candidate cDNAs corresponding to moderately abundant mRNAs were screened by translation of hybrid-selected transcripts in wheat-germ lysates. Translation products were extracted with acetic acid and analyzed by electrophoresis in two dimensions in the presence of unlabeled ribosomal proteins. The identity of translation products that coelectrophoresed with purified ribosomal protein standards was supported by peptide mapping. The cDNAs corresponding to L8 (pL8) and L31 (pL31) hybridized to cytoplasmic mRNAs of 1.4 and 0.9 kb, respectively. In Southern blots of genomic DNA digested with BamHI, HindIII or EcoRI, the cDNA inserts from both pL8 and pL31 gave simple hybridization patterns suggestive of a low copy number for mosquito ribosomal protein genes.

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells.

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.