In vitro reconstitution of Bluetongue virus infectious cores.

Bluetongue virus (BTV) is a vector-borne, nonenveloped icosahedral particle that is organized in two capsids, an outer capsid of two proteins, VP2 and VP5, and an inner capsid (or core) composed of two major proteins, VP7 and VP3, in two layers. The VP3 layer (subcore) encloses viral transcription complex (VP1 polymerase, VP4 capping enzyme, VP6 helicase) and a 10-segmented double-stranded (dsRNA) genome. Although much is known about the BTV capsids, the order of the core assembly and the mechanism of genome packaging remain unclear. Here, we established a cell-free system to reconstitute subcore and core structures with the proteins and ssRNAs, demonstrating that reconstituted cores are infectious in insect cells. Furthermore, we showed that the BTV ssRNAs are essential to drive the assembly reaction and that there is a distinct order of internal protein recruitment during the assembly process. The in vitro engineering of infectious BTV cores is unique for any member of the Reoviridae and will facilitate future studies of RNA-protein interactions during BTV core assembly

Cellular Casein Kinase 2 and Protein Phosphatase 2A Modulate Replication Site Assembly of Bluetongue Virus.

A number of cytoplasmic replicating viruses produce cytoplasmic inclusion bodies or protein aggregates; however, a hallmark of viruses of the Reoviridae family is that they utilize these sites for purposes of replication and capsid assembly, functioning as viral assembly factories. Here we have used bluetongue virus (BTV) as a model system for this broad family of important viruses to understand the mechanisms regulating inclusion body assembly. Newly synthesized viral proteins interact with sequestered viral RNA molecules prior to capsid assembly and double-stranded RNA synthesis within viral inclusion bodies (VIBs). VIBs are predominantly comprised of a BTV-encoded non-structural protein 2 (NS2). Previous in vitro studies indicated that casein kinase 2 (CK2) mediated the phosphorylation of NS2, which regulated the propensity of NS2 to form larger aggregates. Using targeted pharmacological reagents, specific mutation in the viral genome by reverse genetics and confocal microscopy, here we demonstrate that CK2 activity is important for BTV replication. Furthermore, we show that a novel host cell factor, protein phosphatase 2A, is involved in NS2 dephosphorylation and that, together with CK2, it regulates VIB morphology and virus replication. Thus, these two host enzymes influence the dynamic nature of VIB assembly/disassembly, and these concerted activities may be relevant to the assembly and the release of these cores from VIBs

Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate

BACKGROUND: Bluetongue virus (BTV) infection causes haemorrhagic disease in ruminants and induces cell death. The pathogenesis in animals and in cell culture has been linked to BTV-induced apoptosis. RESULTS: In this report, we investigated BTV-induced apoptosis in cell culture in depth and show that both extrinsic (caspase-8 activation) and intrinsic (caspase-9 activation) pathways play roles in BTV apoptosis. Further, by using chemical inhibitors and knock-out cell lines, we show that these pathways act independently of each other in BTV infected cells. In addition to activation of caspase-8, -9 and executioner caspase-3, we also identified that BTV infection causes the activation of caspase-7, which results in the cleavage of poly (ADP-ribose) polymerase (PARP). BTV-induced cell death appears to be due to apoptosis rather than necrosis, as the HMBG-1 was not translocated from the nucleus. We also examined if NF-κB response is related to BTV-induced apoptosis as in reovirus. Our data suggests that NF-κB response is not linked to the induction of apoptosis. It is controlled by the degradation of only IκBα but not IκBβ, resulting in a rapid transient response during BTV infection. This was supported using an NF-κB dependent luciferase reporter gene assay, which demonstrated early response, that appeared to be suppressed by the late stage of BTV replication. Furthermore, virus titres were higher in the presence of NF-κB inhibitor (SN50), indicating that NF-κB has a role in initiating an antiviral environment. In addition, we show that BTV infection induces the translocation of interferon regulatory factors (IRF-3 and IRF-7) into the nucleus. The induction of IRF responses, when measured by IRF dependent luciferase reporter gene assay, revealed that the IRF responses, like NF-κB response, were also at early stage of infection and mirrored the timing of NF-κB induction. CONCLUSION: BTV triggers a wide range of caspase activities resulting in cell apoptosis. Although both NF-κB and IRF responses are induced by BTV infection, they are not sustained

Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.

Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of Epizootic Haemorrhagic Disease Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules

Highly efficient vaccines for Bluetongue virus and a related Orbivirus based on reverse genetics

Bluetongue virus (BTV) reverse genetics (RG), available since 2007, has allowed the dissection of the virus replication cycle, including discovery of a primary replication stage. This information has allowed the generation of Entry-Competent-Replication-Abortive (ECRA) vaccines, which enter cells and complete primary replication but fail to complete the later stage. A series of vaccine trials in sheep and cattle either with a single ECRA serotype or a cocktail of multiple ECRA serotypes have demonstrated that these vaccines provide complete protection against virulent virus challenge without cross-serotype interference. Similarly, an RG system developed for the related African Horse Sickness virus, which causes high mortality in equids has provided AHSV ECRA vaccines that are protective in horses. ECRA vaccines were incapable of productive replication in animals despite being competent for cell entry. This technology allows rapid generation of emerging Orbivirus vaccines and offers immunogenicity and safety levels that surpass attenuated or recombinant routes

Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid.

The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry

Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection.

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication

Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins

BACKGROUND: Studies on Rift Valley Fever Virus (RVFV) infection process and morphogenesis have been hampered due to the biosafety conditions required to handle this virus, making alternative systems such as recombinant virus-like particles, that may facilitate understanding of these processes are highly desirable. In this report we present the expression and characterization of RVFV structural proteins N, Gn and Gc and demonstrate the efficient generation of RVFV virus-like particles (VLPs) using a baculovirus expression system. RESULTS: A recombinant baculovirus, expressing nucleocapsid (N) protein of RVFV at high level under the control of the polyhedrin promoter was generated. Gel filtration analysis indicated that expressed N protein could form complex multimers. Further, N protein complex when visualized by electron microscopy (EM) exhibited particulate, nucleocapsid like-particles (NLPs). Subsequently, a single recombinant virus was generated that expressed the RVFV glycoproteins (Gn/Gc) together with the N protein using a dual baculovirus vector. Both the Gn and Gc glycoproteins were detected not only in the cytoplasm but also on the cell surface of infected cells. Moreover, expression of the Gn/Gc in insect cells was able to induce cell-cell fusion after a low pH shift indicating the retention of their functional characteristics. In addition, assembly of these three structural proteins into VLPs was identified by purification of cells' supernatant through potassium tartrate-glycerol gradient centrifugation followed by EM analysis. The purified particles exhibited enveloped structures that were similar to the structures of the wild-type RVFV virion particle. In parallel, a second recombinant virus was constructed that expressed only Gc protein together with N protein. This dual recombinant virus also generated VLPs with clear spiky structures, but appeared to be more pleomorphic than the VLPs with both glycoproteins, suggesting that Gc and probably also Gn interacts with N protein complex independent of each other. CONCLUSION: Our results suggest that baculovirus expression system has enormous potential to produce large amount of VLPs that may be used both for fundamental and applied research of RVFV

Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly

BACKGROUND: Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function. RESULTS: Our data revealed that expression of full-length NS2 was sufficient for the formation of inclusion bodies (IBs) that were morphologically similar to the VIBs formed during BTV infection. By using either, individual BTV proteins or infectious virions, we found that while the VP3 of the inner capsid (termed as "core") that surrounds the transcription complex was closely associated with both NS2 IBs and BTV VIBs, the surface core protein VP7 co-localized with NS2 IBs only in the presence of VP3. In contrast to the inner core proteins, the outer capsid protein VP2 was not associated with either IBs or VIBs. Like the core proteins, newly synthesized BTV RNAs also accumulated in VIBs. Unlike full-length NS2, neither the amino-, nor carboxyl-terminal fragments formed complete IB structures and each appeared to interfere in overall virus replication when similarly expressed. CONCLUSION: Together, these data demonstrate that NS2 is sufficient and necessary for IB formation and a key player in virus replication and core assembly. Perturbation of NS2 IB formation resulted in reduced virus synthesis and both the N terminal (NS2-1) and C terminal (NS2-2) fragments act as dominant negative mutants of NS2 function

Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress

BACKGROUND: The VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. RESULTS: In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. CONCLUSION: The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells