Publications

The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-beta), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-beta, IRF-3. N protein dramatically inhibited expression from an NF-kappaB-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.

Mouse hepatitis virus (MHV) was used as a model to study the interaction of coronaviruses with the alpha/beta interferon (IFN-alpha/beta) response. While MHV strain A59 appeared to induce IFN-beta gene transcription and low levels of nuclear translocation of the IFN-beta transcription factor interferon regulatory factor 3 (IRF-3), MHV did not induce IFN-beta protein production during the course of infection in L2 mouse fibroblast cells. In addition, MHV was able to significantly decrease the level of IFN-beta protein induced by both Newcastle disease virus (NDV) and Sendai virus infections, without targeting it for proteasomal degradation and without altering the nuclear translocation of IRF-3 or IFN-beta mRNA production or stability. These results indicate that MHV infection causes an inhibition of IFN-beta production at a posttranscriptional level, without altering RNA or protein stability. In contrast, MHV induced IFN-beta mRNA and protein production in the brains of infected animals, suggesting that the inhibitory mechanisms observed in vitro are not enough to prevent IFN-alpha/beta production in vivo. Furthermore, MHV replication is highly resistant to IFN-alpha/beta action, as indicated by unimpaired MHV replication in L2 cells pretreated with IFN-beta. However, when L2 cells were coinfected with MHV and NDV in the presence of IFN-beta, NDV, but not MHV, replication was inhibited. Thus, rather than disarming the antiviral activity induced by IFN-beta pretreatment completely, MHV may be inherently resistant to some aspects of the antiviral state induced by IFN-beta. These findings show that MHV employs unique strategies to circumvent the IFN-alpha/beta response at multiple steps.

Effective delivery systems are needed to design efficacious vaccines against the obligate intracellular bacterial pathogen, Chlamydia trachomatis. Potentially effective delivery vehicles should promote the induction of adequate levels of mucosal T-cell and antibody responses that mediate long-term protective immunity. Antigen targeting to the nasal-associated lymphoid tissue (NALT) is effective for inducing high levels of specific immune effectors in the genital mucosa, and therefore suitable for vaccine delivery against genital chlamydial infection. We tested the hypothesis that live attenuated influenza A viruses are effective viral vectors for intranasal delivery of subunit vaccines against genital chlamydial infection. Recombinant influenza A/PR8/34 (H1N1) viruses were generated by insertion of immunodominant T-cell epitopes from chlamydial major outer membrane protein into the stalk region of the neuraminidase gene. Intranasal immunization of mice with viral recombinants resulted in a strong T helper 1 (Th1) response against intact chlamydial elementary bodies. Also, immunized mice enjoyed a significant state of protective immunity (P > 0.002) by shedding less chlamydiae and rapidly clearing the infection. Furthermore, a high frequency of Chlamydia-specific Th1 was measured in the genital mucosal and systemic draining lymphoid tissues within 24 hr after challenge of vaccinated mice. Moreover, multiple epitope delivery provided a vaccine advantage over single recombinants. Besides, long-term protective immunity correlated with the preservation of a robustly high frequency of specific Th1 cells and elevated immunoglobulin G2a in genital secretions. Because live attenuated influenza virus vaccines are safe and acceptable for human use, they may provide a new and reliable approach to deliver efficacious vaccines against sexually transmitted diseases.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.

The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-alpha/beta) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) . poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) . poly(rC), poly(rA) . poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-beta promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKepsilon and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

It was recently shown that the 7a protein of severe acute respiratory syndrome coronavirus induces biochemical changes associated with apoptosis. In this study, the mechanism by which the 7a protein induces apoptosis was examined. The 7a protein was tested for the ability to inhibit cellular gene expression because several proapoptotic viral proteins with this function have previously been identified. 7a protein inhibited expression of luciferase from an mRNA construct that specifically measures translation, whereas inhibitors of transcription and nucleocytoplasmic transport did not. The inhibition of translation and other cellular processes of gene expression have been associated with the induction of a stress response in cells. Western blot analysis using phosphospecific antibodies indicated that 7a protein activated p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal protein kinase/stress-activated protein kinase. Taken together, these data indicate that the induction of apoptosis by the 7a protein may be related to its ability to inhibit cellular translation and activate p38 MAPK.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-beta) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. We and others have previously shown that the replication of SARS-CoV can be suppressed by exogenously added interferon (IFN), a cytokine which is normally synthesized by cells as a reaction to virus infection. Here, we demonstrate that SARS-CoV escapes IFN-mediated growth inhibition by preventing the induction of IFN-beta. In SARS-CoV-infected cells, no endogenous IFN-beta transcripts and no IFN-beta promoter activity were detected. Nevertheless, the transcription factor interferon regulatory factor 3 (IRF-3), which is essential for IFN-beta promoter activity, was transported from the cytoplasm to the nucleus early after infection with SARS-CoV. However, at a later time point in infection, IRF-3 was again localized in the cytoplasm. By contrast, IRF-3 remained in the nucleus of cells infected with the IFN-inducing control virus Bunyamwera delNSs. Other signs of IRF-3 activation such as hyperphosphorylation, homodimer formation, and recruitment of the coactivator CREB-binding protein (CBP) were found late after infection with the control virus but not with SARS-CoV. Our data suggest that nuclear transport of IRF-3 is an immediate-early reaction to virus infection and may precede its hyperphosphorylation, homodimer formation, and binding to CBP. In order to escape activation of the IFN system, SARS-CoV appears to block a step after the early nuclear transport of IRF-3.

The Thogoto virus (THOV) is a member of the family Orthomyxoviridae. It prevents induction of alpha/beta interferons (IFN) in cell culture and in vivo via the action of the viral ML protein. Phenotypically, the effect of THOV ML resembles that of the NS1 protein of influenza A virus (FLUAV) in that it blocks the expression of IFN genes. IFN expression depends on IFN regulatory factor 3 (IRF3). Upon activation, IRF3 forms homodimers and accumulates in the nucleus where it binds the transcriptional coactivator CREB-binding protein (CBP). Here, we show that expression of ML blocked the transcriptional activity of IRF3 after stimulation by virus infection. Further biochemical analysis revealed that ML acts by blocking IRF3 dimerization and association with CBP. Surprisingly, however, ML did not interfere with the nuclear transport of IRF3. Thus, the action of ML differs strikingly from that of FLUAV NS1 that prevents IFN induction by retaining IRF3 in the cytoplasm.

The reactivity of a panel of 12 monoclonal antibodies raised against the human respiratory syncytial virus 22 kDa (22K) protein was tested by Western blotting with a set of 22K deletion mutants. The results obtained identified sequences in the C-terminal half of the 22K polypeptide required for integrity of most antibody epitopes, except for epitope 112, which was lost in mutants with short N-terminal deletions. This antibody, in contrast to the others, failed to immunoprecipitate the native 22K protein, indicating that the N terminus of this protein is buried in the native molecule and exposed only under the denaturing conditions of Western blotting. In addition, N-terminal deletions that abolished reactivity with monoclonal antibody 112 also inhibited phosphorylation of the 22K protein previously identified at Ser-58 and Ser-61, suggesting that the N terminus is important in regulating the 22K protein phosphorylation status, most likely as a result of its requirement for protein folding.