Publications

The tick-transmitted orthomyxovirus Thogoto virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.

The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins.

BACKGROUND: Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals.

RESULTS: Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.

CONCLUSIONS: Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.

Newcastle disease virus (NDV) has been previously shown to possess oncolytic activity, causing specific lysis of cancerous but not normal cells. Here we show that despite these findings, the oncolytic efficiency of naturally occurring NDV strains can still be relatively low, as many tumors exhibit strong innate immune responses that suppress viral replication and spread. To overcome this problem, we generated a recombinant fusogenic NDV expressing influenza NS1 protein, a protein exhibiting interferon (IFN)-antagonist and antiapoptotic functions in human and mouse cells. Interestingly, the resultant virus was dramatically enhanced in its ability to form syncytia, lyse a variety of human and mouse tumor cell lines, and suppressed the induction of the cellular IFN responses. Using the aggressive syngeneic murine melanoma model, we show that the NDV-NS1 virus is more effective than virus not expressing NS1 in clearing the established footpad tumors and results in higher overall long-term animal survival. In addition, mice treated with NDV-NS1 exhibited no signs of toxicity to the virus and developed tumor-specific cytotoxic T lymphocyte (CTL) responses. These findings demonstrate that modulation of innate immune responses by NDV results in enhancement of its oncolytic properties and warrant further investigation of this strategy in design of oncolytic NDV vectors against human tumors.

Lymphocytic choriomeningitis virus (LCVM) nucleoprotein (NP) counteracts the host type I interferon (IFN) response by inhibiting activation of the IFN regulatory factor 3 (IRF3). In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.

ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L(-/-) and ISG15(-/-) mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.

SARS coronavirus (SARS-CoV) is known to efficiently suppress the induction of antiviral type I interferons (IFN-alpha/beta) in non-lymphatic cells through inhibition of the transcription factor IRF-3. Plasmacytoid dendritic cells, in contrast, respond to infection with production of high levels of IFNs. Here, we show that pretreatment of non-lymphatic cells with small amounts of IFN-alpha (IFN priming) partially overturns the block in IFN induction imposed by SARS-CoV. IFN priming combined with SARS-CoV infection substantially induced genes for IFN induction, IFN signalling, antiviral effector proteins, ubiquitination and ISGylation, antigen presentation and other cytokines and chemokines, whereas each individual treatment had no major effect. Curiously, however, despite this typical IFN response, neither IRF-3 nor IRF-7 was transported to the nucleus as a sign of activation. Taken together, our results suggest that (i) IFN, as it is produced by plasmacytoid dendritic cells, could enable tissue cells to launch a host response to SARS-CoV, (ii) IRF-3 and IRF-7 may be active at subdetectable levels, and (iii) SARS-CoV does not activate IRF-7.

Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR's antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5'-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1's dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses.

A hallmark of immune activation by certain RNA sequences is the generation of interferon responses. However, the study of immunostimulatory RNA (isRNA) has been hindered by costly and slow methods, particularly in vitro. We have developed a cell-based assay to detect human type I interferon (IFN) that reliably senses both IFN-alpha and IFN-beta simultaneously. The human 293T cell line was stably transfected with a fusion gene of monomeric red fluorescent protein (mRFP) under the transcriptional control of an interferon-stimulated response element (ISRE). High levels of mRFP are expressed following activation of the type I IFN receptor (IFNAR). Using this method, detection limits for IFN similar to that of ELISA can be achieved but with a greater dynamic range and in a high-throughput manner. As a proof of concept, we utilized this method to screen a library of cationic lipid-like materials that form nanoparticle complexes with RNA for induction of innate immune responses in vitro. We expect the screening and detection methods described herein may provide a useful tool in elucidating mechanisms that govern the delivery of RNA molecules to effector cells and receptors of the innate immune system. We apply this tool to investigate isRNA drug delivery, but it may also find use in other applications for which high-throughput detection of type 1 IFN is desired.