Richard M. Elliott

Molecular biology of the bunyaviridae

Introduction. More than 300 viruses, mostly arthropod-transmitted, are classified into the family Bunyaviridae, making it one of the largest groupings of animal viruses (Karabatsos, 1985). Until relatively recently these viruses were somewhat the ‘Cinderellas’ of animal virology, but with the increased recognition of their role in human diseases together with the results generated by the application of molecular techniques, the Bunyaviridae have achieved greater respectability. Rift Valley fever, Crimean-Congo haemorrhagic fever and California encephalitis viruses are serious human pathogens that are classified in the family Bunyaviridae. In the tropics febrile illnesses are often diagnosed under the ‘great umbrella’ (Downs, 1975) of malaria and treated as such; in fact many cases are probably caused by members of the Bunyaviridae, although true diagnosis is rarely achieved (Shope, 1985). Hantaan and related viruses, the causative agents of haemorrhagic fever with renal syndrome, are now recognized as belonging to the Bunyaviridae (Schmaljohn & Dalrymple, 1983) and cause a severe haemorrhagic disease with significant mortality throughout Asia, especially in China.

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East

Hepatitis C virus (HCV) type 4 is the predominant genotype found throughout the Middle East and parts of Africa, often in association with high population prevalence as in Egypt. To investigate more fully its evolutionary relationship with other genotypes of HCV, and to study its overall genome organization, we have determined the entire sequence encompassing the coding region of the genotype 4a isolate ED43, obtained from an HCV-infected individual from Egypt. The sequence of ED43 contained a single open reading frame encoding a polyprotein of 3008 amino acids (aa), smaller than that reported for other HCV genotypes which vary from 3010 aa to 3037 aa. The nucleotide and amino acid sequences were compared with the full-length sequences already reported for genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b and those of isolates JKO49 and JKO46 described as types 10a and 11a. The differences in length of the polyprotein originated in variable regions in the E2 and NS5A genes. The complete sequence of ED43 confirmed the classification of type 4 as a separate major genotype.

NSs Protein of Rift Valley Fever Virus Induces the Specific Degradation of the Double-Stranded RNA-Dependent Protein Kinase

ABSTRACT Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-α/β]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

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.

Optineurin Negatively Regulates the Induction of IFNβ in Response to RNA Virus Infection

The innate immune response provides a critical defense against microbial infections, including viruses. These are recognised by pattern recognition receptors including Toll-like receptors (TLRs) and RIG-I like helicases (RLHs). Detection of virus triggers signalling cascades that induce transcription of type I interferons including IFNβ, which are pivotal for the initiation of an anti-viral state. Despite the essential role of IFNβ in the anti-viral response, there is an incomplete understanding of the negative regulation of IFNβ induction. Here we provide evidence that expression of the Nemo-related protein, optineurin (NRP/FIP2), has a role in the inhibition of virus-triggered IFNβ induction. Over-expression of optineurin inhibited Sendai-virus (SeV) and dsRNA triggered induction of IFNβ, whereas depletion of optineurin with siRNA promoted virus-induced IFNβ production and decreased RNA virus replication. Immunoprecipitation and immunofluorescence studies identified optineurin in a protein complex containing the antiviral protein kinase TBK1 and the ubiquitin ligase TRAF3. Furthermore, mutagenesis studies determined that binding of ubiquitin was essential for both the correct sub-cellular localisation and the inhibitory function of optineurin. This work identifies optineurin as a critical regulator of antiviral signalling and potential target for future antiviral therapy.

RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment

ABSTRACT The genome of Bunyamwera virus (BUN) (familyBunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5′-terminal 32 and 3′-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments.

ABSTRACT The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.

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.

RNA Polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.