Bunyamwera Virus Research Articles

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

Molecular characterization of African orthobunyaviruses

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5-15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination

Messenger RNA transcription by Bunyaviridae family members is unique within the group of negative-strand RNA viruses as it requires on-going protein synthesis. The long-standing model explaining this phenomenon proposes that the translational requirement is not for a protein product, but instead is for ribosomal translocation along nascent mRNAs. This movement is proposed to disrupt spurious transcription termination signals that otherwise cause premature mRNA truncation leading to a fatal loss of gene expression. This model was tested by introducing translational stop codons into model RNA genomes of Bunyamwera virus, the prototypic member of the Bunyaviridae family. This directly showed that translation of nascent mRNAs prevents transcription termination. While such coupling of transcription and translation is common in prokaryotic systems, these results represent the first report of such obligatory coupling in a eukaryotic cell environment. The results also provide insight into the bunyavirus termination mechanism and suggest it is mechanistically similar to prokaryotic intrinsic termination.

Interaction of Bunyamwera Orthobunyavirus NSs Protein with Mediator Protein MED8: a Mechanism for Inhibiting the Interferon Response

The NSs protein of Bunyamwera virus (Bunyaviridae) is an antiapoptotic interferon antagonist involved in silencing host protein expression by interfering with mRNA synthesis. Here, we show that the ability to inhibit both host transcription and the interferon response is linked to interaction of NSs with the MED8 component of Mediator, a protein complex necessary for mRNA production. The interacting domain on NSs was mapped to the C-terminal region, which contains amino acids conserved among orthobunyavirus NSs proteins. A recombinant virus in which the interacting domain in NSs was deleted had strongly reduced ability to inhibit host protein expression and was unable to inhibit the interferon response. This study provides further information on the mechanisms by which bunyavirus nonstructural proteins are involved in pathogenesis.

Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

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.

Genetic characterization of Batai virus indicates a genomic reassortment between orthobunyaviruses in nature

Two viruses were isolated from bovine blood in the southernmost part of Japan in 1994 and 2001, respectively. Genetic analyses showed that the viruses were Batai virus of the genus Orthobunyavirus of the family Bunyaviridae. The sequencing of three genomic RNA segments of the Japanese and Malaysian Batai virus strains revealed that the M RNA segment of Batai virus had high sequence identity with that of Ngari virus. Our results indicate that Ngari virus is a genetic reassortant with S and L RNA segments from Bunyamwera virus and an M RNA segment from Batai virus.

Batai and Ngari Viruses: M Segment Reassortment and Association with Severe Febrile Disease Outbreaks in East Africa

Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.

Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera virus

Bunyamwera virus (BUNV) is the prototypic member of the Bunyaviridae family of segmented negative-sense RNA viruses. The BUNV nucleocapsid protein has been cloned and expressed in Escherichia coli. The purified protein has been crystallized and a complete data set has been collected to 3.3 angstroms resolution at a synchrotron source. Crystals of the nucleocapsid protein belong to space group C2, with unit-cell parameters a = 384.7, b = 89.8, c = 89.2 angstroms, beta = 94.4 degrees . Self-rotation function analysis of the X-ray diffraction data has provided insight into the oligomeric state of the protein as well as the orientation of the oligomers in the asymmetric unit. The structure determination of the protein is ongoing.

Identification of the Bunyamwera bunyavirus transcription termination signal

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3' ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5' terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3'-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5' non-translated region (NTR) of the S segment. A 6 nt region (3'-GUCGAC-5') within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3'-UGUCG-5', that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5' NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.

Genetic elements regulating packaging of the Bunyamwera orthobunyavirus genome

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) comprises three segments of negative-sense, single-stranded RNA. The RNA segments are encapsidated by the viral nucleocapsid (N) protein and form panhandle-like structures through interaction of complementary sequences at their 5' and 3' termini. Transcription and replication of a BUN genome analogue (minireplicon), comprising the viral non-coding sequences flanking a reporter gene, requires just the viral RNA polymerase (L protein) and N protein. Here, sequences of Bunyamwera serogroup M segment RNAs were compared and conserved elements within nt 20-33 of the 3' and 5' non-coding regions that can affect packaging of minireplicons into virions were identified. RNA-folding models suggest that a conserved sequence within nt 20-33 of the 5' end of the genome segments maintains conserved structural features necessary for efficient transcription. Competitive packaging experiments using M, L and S segment-derived minireplicons that encode different reporter genes showed variable packaging efficiencies of the three segments. Packaging of a particular segment appeared to be independent of the presence of other segments and, for the S segment, packaging efficiency was unaffected by the inclusion of viral coding sequences in the minireplicon.

Role of N-Linked Glycans on Bunyamwera Virus Glycoproteins in Intracellular Trafficking, Protein Folding, and Virus Infectivity

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-beta-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.

Homotypic interaction of Bunyamwera virus nucleocapsid protein.

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein.

The Bunyamwera virus mRNA transcription signal resides within both the 3' and the 5' terminal regions and allows ambisense transcription from a model RNA segment.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of RNA viruses. BUNV genomic strands are templates for both replication and transcription, whereas the antigenomic RNAs serve only as templates for replication. By mutagenesis of model templates, we showed that the BUNV transcription promoter comprises elements within both the 3' and the 5' nontranslated regions. Using this information, we designed a model ambisense BUNV segment that transcribed BUNV S mRNA from the genomic strand and green fluorescent protein (GFP) mRNA from the antigenome. Demonstration of GFP expression showed that this ambisense strategy represents a viable approach for generating BUNV segments able to express additional proteins.

Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus

Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-beta-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.

Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

Role of the conserved nucleotide mismatch within 3'- and 5'-terminal regions of Bunyamwera virus in signaling transcription.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3' and 5' nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable. Here, we investigate the basis for the fundamentally different signaling abilities of genomic and antigenomic strands. We show that the identity of only nucleotide position 9 within the genomic 3' NTR is critical for the different RNA synthesis characteristics of genomic and antigenomic strands, thus identifying this nucleotide as an essential component of the transcription promoter. This nucleotide is distinctive, as it interrupts an unbroken run of conserved complementary nucleotides within the 3' and 5' NTRs of all three segments. Our results show that the conserved mismatched arrangement of this nucleotide plays no detectable role in signaling transcription. Instead, we show that the transcription-signaling ability of this position is entirely dependent on its nucleotide identity. We further show that while a U residue at 3' position 9 is strongly preferred for transcription activity in the context of the genomic promoter, it does not signal transcription in the context of the antigenomic promoter. Therefore, our results show that the identity of 3' position 9 is crucial for signaling BUNV transcription; however, it is not the sole determinant.

Efficient bunyavirus rescue from cloned cDNA

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.

Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins.

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN; family Bunyaviridae) accumulate in the Golgi complex, where virion maturation occurs. The Golgi targeting and retention signal has previously been shown to reside within the Gn protein. A series of truncated Gn and glycoprotein precursor cDNAs were constructed by progressively deleting the coding region of the transmembrane domain (TMD) and the cytoplasmic tail. We also constructed chimeric proteins of BUN Gc, enhanced green fluorescent protein (EGFP), and human respiratory syncytial virus (HRSV) fusion (F) protein that contain the Gn TMD with various lengths of its adjacent cytoplasmic tails. The subcellular localization of mutated BUN glycoproteins and chimeric proteins was investigated by double-staining immunofluorescence with antibodies against BUN glycoproteins or the HRSV F protein and with antibodies specific for the Golgi complex. The results revealed that Gn and all truncated Gn proteins that contained the intact TMD (residues 206 to 224) were able to translocate to the Golgi complex and also rescued the Gc protein, which is retained in the endoplasmic reticulum when expressed alone, to this organelle. The rescued Gc proteins acquired endo-beta-N-acetylglucosaminidase H resistance. The Gn TMD could also target chimeric EGFP to the Golgi and retain the F protein, which is characteristically expressed on the surface of HRSV-infected cells, in the Golgi. However, chimeric BUN Gc did not translocate to the Golgi, suggesting that an interaction with Gn is involved in Golgi retention of the Gc protein. Collectively, these data demonstrate that the Golgi targeting and retention signal of BUN glycoproteins resides in the TMD of the Gn protein.

Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa.

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.