Anne Bridgen

Ovarian Tumor Domain-Containing Viral Proteases Evade Ubiquitin- and ISG15-Dependent Innate Immune Responses

Summary Ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) reversibly conjugate to proteins and mediate important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial, and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases from nairoviruses and arteriviruses, two unrelated groups of RNA viruses, hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. Expression of a viral OTU domain-containing protein antagonizes the antiviral effects of ISG15 and enhances susceptibility to Sindbis virus infection in vivo. We also show that viral OTU domain-containing proteases inhibit NF-κB-dependent signaling. Thus, the deconjugating activity of viral OTU proteases represents a unique viral strategy to inhibit Ub- and ISG15-dependent antiviral pathways.

Bunyamwera Bunyavirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon

ABSTRACT Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera virus.

Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence.

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5′ end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.

Bunyamwera Virus Nonstructural Protein NSs Counteracts Interferon Regulatory Factor 3-Mediated Induction of Early Cell Death

ABSTRACT The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.

Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs

ABSTRACT Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.

Preliminary Characterization of the Alcelaphine Herpesvirus 1 Genome

Alcelaphine herpesvirus type 1 (AHV-1) is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in deer and cattle. The genomes of the attenuated WC11 isolate and the virulent C500 isolate have been studied. The genome of WC11 comprises a region of unique DNA of approximately 130 kbp, which has a G + C content of 50%, and approximately 30 kbp of additional tandem direct repeat sequences with G + C content of 72%. WC11 possesses a major repeat sequence of 950 bp interspersed with a small number of related sequences of different length; these sequences are probably terminal in location. DNA from the C500 isolate has a similar restriction profile to that of WC11 in the unique region, but only one repeat sequence of 1050 bp is present. We propose, on the basis of biological and structural properties, that AHV-1 be included within the gamma 2 group of herpesviruses of which herpesvirus ateles is the prototype.

Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU.

Dugbe virus ovarian tumour domain interferes with ubiquitin/ISG15-regulated innate immune cell signalling

The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-α/NF-κB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-α-induced activation of NF-κB.

Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.