No abstract

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

Bunyaviruses are trisegmented, negative-sense RNA viruses. Previously, we described a rescue system to recover infectious Bunyamwera virus (genus Orthobunyavirus) entirely from cloned cDNA (Bridgen, A. and Elliott, R.M. (1996) Proc. Nat. Acad. Sci. USA 93, 15400-15404) utilizing a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase to drive intracellular transcription of transfected T7 promoter-containing plasmids. Here we report efforts to improve the efficiency of the system by comparing different methods of providing T7 polymerase. We found that a BHK-derived cell line BSR-T7/5 that constitutively expresses T7 RNA polymerase supported efficient and reproducible recovery of Bunyamwera virus, routinely generating >10(7) pfu per rescue experiment. Furthermore, we show that the virus can be recovered from transfecting cells with just three plasmids that express full-length antigenome viral RNAs, greatly simplifying the procedure. We suggest that this procedure should be applicable to viruses in other genera of the family Bunyaviridae and perhaps also to arenaviruses.

Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G 0 and early G 1 , but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid-to late G 1 phase, when RB becomes phosphorylated by cyclin D-and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.The retinoblastoma protein RB is a highly abundant tumor suppressor that can bind and regulate a variety of transcription factors (reviewed in Refs. 1-4). One example that has been added recently to the growing list of RB-binding proteins is the RNA polymerase (pol) 1 III-specific factor TFIIIB (5, 6). Recombinant RB was shown to bind to TFIIIB in vitro and repress its activity (5, 6). Furthermore, coimmunoprecipitation and cofractionation experiments demonstrated a stable association between endogenous cellular RB and TFIIIB (6). The functional significance of this interaction was shown in studies of RB knockout mice, since primary fibroblasts from RbϪ/Ϫ mice display elevated TFIIIB activity relative to fibroblasts derived from their wild-type siblings (6). These results establish TFIIIB as a bona fide target for repression by RB. Similar approaches have shown that TFIIIB is also bound and repressed by the RB-related proteins p107 and p130 (7).TFIIIB is required for the expression of all pol III templates (reviewed in Refs. 8 and 9). It serves to recruit the polymerase to a promoter and position it over the transcription start site (10). By interacting with this general factor, RB appears able to regulate the expression of all pol III-transcribed genes, including tRNA, 5 S rRNA, U6 small nuclear RNA, VA1, and Alu genes (5, 6, 11). Since a high rate of tRNA and rRNA synthesis is required to sustain rapid growth, it has been speculated that the inhibition of pol III transcription may contribute to the growth suppression capacity of RB (12)(13)(14).RB function is regulated by cyclin-dependent kinases (reviewed in Refs. 2 and 3 and Ref. 15). The cyclin D-dependent kinases CDK4 and CDK6 phosphorylate RB partially and the process is completed by cyclin E-CDK2 (16, 17). The action of cyclin E-CDK2 appears to depend on prior phosphorylation by the cyclin D-dependent kinases (17). At least 10 serine and threonine residues can become phosp...

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein–protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

Viruses in the genus

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb ؊/؊ p130 ؊/؊ fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.