Biochemical and serological comparisons of Australian bunyaviruses belonging to the Simbu serogroup.

Comparative analysis of the structural and possible non-structural proteins of seven Simbu serogroup bunyaviruses isolated in Australia revealed them all to be similar in size to those of Bunyamwera virus, the prototype of the Bunyavirus genus. The molecular weights of the structural proteins for these bunyaviruses (Akabane, Aino, Tinaroo, Douglas, Peaton, Facey's Paddock and Thimiri viruses) were 193K to 205K (L), 103K to 125K (G1), 33K to 37K (G2) and 25K to 26K (N). Analysis of the virion RNA of three viruses (Akabane, Douglas and Facey's Paddock) showed them all to be similar to Bunyamwera virus RNA, apparent Mr values being 2.6 X 10(6) (L), 1.4 X 10(6) to 1.9 X 10(6) (M) and 0.24 X 10(6) to 0.42 X 10(6) (S). Host cell protein synthesis was switched off late during infection, revealing four structural proteins L, G1, G2 and N. Comparative analysis of these protein profiles in infected Vero cells showed each virus, although similar, to be unique and easily identified; this method of comparison was efficient and rapid compared to the difficulty in obtaining adequate amounts of purified virus for analysis. Additionally, for all viruses except Douglas, two to four possible non-structural proteins were identified, with an Mr range from 12K to 30K. The viruses Akabane and Tinaroo, which have previously been shown to cross-react by plaque inhibition virus neutralization tests, were readily distinguished in migration of the G1 glycoprotein and by analysis of plaque reduction virus neutralization data using linear regression analysis of the dose-response curves. Using these same analyses, the differences between Aino and Douglas viruses, also related by plaque inhibition, were even greater. Application of the biochemical analysis of virus-specified proteins and some serological comparisons identified a mixed pool of different viruses in two unknown isolates grouped as Simbu serogroup viruses, and further identified a potential teratogenic strain in one of the two pools.

Some physical and chemical properties of Bhanja virus.

Bhanja virus is acid-labile, relatively thermostable, resistant to trypsin and heparin; a complete inactivation was achieved with chloramine B or formaldehyde, while phenol was ineffective, and UV radiation only partially effective.

The proteins and RNAs specified by Clo Mor virus, a Scottish Nairovirus.

The proteins and RNAs of Clo Mor virus have been analysed. Virus-specific proteins in infected cells had previously been identified by isotopic labelling and radioimmunoprecipitation; these were three glycoproteins (mol. wt. 115K, 90K and 80K) and an unglycosylated nucleocapsid (N) protein (50K). We have performed pulse-chase experiments which indicated that the 115K protein is processed to give the 90K and 80K proteins, while a 45K protein was detected in released virions after prolonged chase. Translation in vitro of mRNA extracted from Clo Mor virus-infected cells resolved only the N protein. Three species of RNA were extracted from Clo Mor virus intracellular nucleocapsids and have been designated L (11000 to 13000 bases), M (6300 bases) and S (1900 bases). The processing of viral proteins and the sizes of RNAs are characteristic of the Nairovirus genus of the family Bunyaviridae.

The proteins and RNAs of St. Abb's Head virus, a Scottish Uukuvirus.

The proteins and RNAs of St. Abb's Head virus have been analysed, and were found to be characteristic of the Uukuvirus genus of the family Bunyaviridae. Two glycoproteins, G1 and G2 (mol. wt. 62K and 75K), and a nucleocapsid protein, N (mol. wt. 25K), were detected in infected cells by immunoprecipitation; the synthesis of N preceded the synthesis of G1 and G2. The glycoproteins were relatively cysteine-rich compared to the N protein, and the unglycosylated forms of G1 and G2 (using the inhibitor tunicamycin) had a mol. wt. of about 58K. Translation in vitro of mRNA from infected cells gave two immunoprecipitable products which are thought to be equivalent to N and G1/G2. Three RNA species were found in St. Abb's Head virus nucleocapsids, and were estimated to be 8500 bases (L), 3600 bases (M) and 1900 bases (S) in length. At least one additional virus-specific RNA species was detected in infected cells. The similarity between the proteins and RNAs of St. Abb's Head virus and Uukuniemi virus (the prototype of the genus) is discussed.

Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopus laevis.

The XTC-2 cell line, derived from Xenopus laevis, supported the replication of representative viruses from each of the four genera in the family Bunyaviridae. Generally, viral titres were higher in XTC-2 cells than in other susceptible cell lines, and for some viruses plaques were detected earlier in XTC-2 cells. The XTC-2 cell line permitted comparative analyses of bunyavirus-specific protein synthesis. The patterns of synthesis of viral proteins, characteristic of each of the genera, were observed with representative viruses. These studies provided biochemical characterization of two Scottish isolates, which support the inclusion of Clo Mor virus in the Nairovirus genus and St Abb's Head (M349) virus in the Uukuvirus genus.

La Crosse virus G1 glycoprotein undergoes a conformational change at the pH of fusion.

La Crosse virus, a member of the family Bunyaviridae, can mediate cell-to-cell fusion after mild acidification. Either of its two envelope glycoproteins could potentially be responsible for this function. The large glycoprotein (G1) undergoes a conformational change at the pH of activation of the fusion function, resulting in both an alteration in the cleavage pattern produced by amino-acid-specific proteases, and in a change in its antigenicity, as defined by altered binding of monoclonal antibodies.

Hantaan virus: identification of virion proteins.

SDS-PAGE and immunoprecipitation analyses were carried out on the virion and cell-associated proteins of Hantaan virus, the causative agent of haemorrhagic fever with renal syndrome (HFRS). Purified virions have a density of 1.17 g/ml in sucrose, and contain four proteins with molecular weights of 45 000 (45K), 56K, 72K and 200K, confirming recent evidence that the virus is a member of the family Bunyaviridae. Detergent treatment of virions indicates that the 45K protein is the virus nucleoprotein. Both the 72K and the 56K proteins were labelled with [3H]glucosamine and were removed from virions by bromelain treatment, indicating that they are envelope glycoproteins. The 200K protein was found only in [35S]methionine-labelled preparations. By analogy to prototype viruses of the family Bunyaviridae, these proteins were designated N, G1, G2, and L respectively. Three virus-specific proteins (N, G1, G2) were detected in virus-infected cells. These proteins were precipitable by human convalescent serum and by serum of a Rattus norvegicus trapped in the United States. No additional virus proteins were detected in infected cells. These results confirm recent morphological and RNA studies that Hantaan virus is a member of the family Bunyaviridae. Our results also support the suggestion that Hantaan virus be placed in a new genus of Bunyaviridae.

Localized conserved regions of the S RNA gene products of bunyaviruses are revealed by sequence analyses of the Simbu serogroup Aino virus.

The complete nucleotide sequence has been determined for the S RNA of Aino virus, a member of the Simbu serogroup (Bunyavirus genus, family Bunyaviridae). The S RNA is 850 nucleotides long (2.76 X 10(5) daltons) and in the viral complementary sequence has a short 5' non-coding region of 34 nucleotides and a more extensive 3' non-coding region of 117 nucleotides. The 3'-5' complementarity of the Aino S RNA is about 25 residues long, depending on the arrangement. The Aino sequence predicts that, like snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses (Bishop, D.H.L., et al. (1982) Nucleic Acids Res., 10, 3703-3713; Akashi, H. and Bishop, D.H.L. (1983) J. Virol. 45, 1155-1158), there are two S coded gene products, a nucleoprotein N, and a non-structural protein, NSS, that are read from overlapping reading frames in the viral complementary sequence. The Aino N primary gene product is composed of 233 amino acids (26.2 X 10(3) daltons) and is 45% homologous in sequence with that of LAC virus. The NSS protein of Aino virus is composed of 91 amino acids (10.5 X 10(3) daltons) and is 35% homologous in sequence with the LAC NSS protein. Unlike those viruses there are no uridylate tracts longer than 4 residues in the 5' non-coding region of the S viral RNA that could function as a template for polyadenylation of Aino S mRNA species.

Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae.

Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, was examined for nucleic acid characteristics which would support its previously proposed inclusion in the virus family Bunyaviridae. Nucleocapsid RNA from Hantaan virions and a control bunyavirus were examined for ribonuclease A (RNase A) sensitivity. Both viruses exhibited a similar accessibility of RNA within nucleocapsids to digestion by RNase A. Complete digestion of the RNA of both viruses was affected with high concentrations of ribonuclease. Evidence for negative strand RNA polarity was obtained by an in vitro transcriptase assay. RNA dependent RNA polymerase activity was associated with Hantaan virions. Polymerase activity required manganese and nucleoside triphosphates and was enhanced by magnesium, 2-mercaptoethanol, and sodium chloride. Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA. A common 3' terminal sequence of the three genome segments was determined to be 3' AUCAUCAUCUG. This sequence is different from those reported for viruses within the four recognized genera of the Bunyaviridae. Because all other data were consistent with nucleic acid characteristics of the Bunyaviridae, we propose a separate genus within the Bunyaviridae with Hantaan as its prototype virs.

Identification of four complementary RNA species in Akabane virus-infected cells.

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 X 10(6), 1.5 X 10(6), and 0.48 X 10(6) for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind to oligodeoxythymidylate-cellulose. Kinetic analysis of cRNA synthesis in infected cells at various times postinfection suggested that cRNA synthesis could be detected as early as 2 h postinfection and that maximal synthesis occurred at 4 to 6 h postinfection. The RNAs synthesized in infected cells could be partially resolved by sucrose density gradient centrifugation. The RNA fraction that cosedimented with the S segment of viral genomic RNA yielded two duplex RNA species when hybridized with viral genomic RNA, suggesting the presence of two small cRNA species. Specific hybridization with individual viral genomic RNAs confirmed that two species of cRNA are coded by the S RNA segment. Analysis of cRNA synthesis in the presence of the protein synthesis inhibitors cycloheximide and puromycin indicated that cycloheximide completely inhibited virus-specific RNA synthesis early and late in infection, whereas a very low level of synthesis occurred in the presence of puromycin. The inhibitory effects of these drugs were found to be reversible when the drugs were washed from the cells. It is concluded that continued protein synthesis is required for cRNA synthesis to proceed in Akabane virus-infected cells.

[Virus--the causative agent of hemorrhagic fever with renal syndrome--a new representative of the family Bunyaviridae]. / Virus--vozbuditel' gemorragicheskoi likhoradki s pochechnym sindromom--novyi predstavitel' semeistva Bunyaviridae.

Physicochemical characteristics (sedimentation coefficient, buoyant density of particles, as well as ribonuclein of these particles) and morphology of the causative agent of hemorrhagic fever with renal syndrome were found to be similar to those of the other members of the Bunyaviridae family.

Characterization of Leanyer virus: resemblance to Bunyavirus.

The properties of Leanyer virus, isolated in Northern Australia in 1974, were compared with those of Bunyamwera virus. Leanyer virus replicated in BHK-21 and Vero cells. In sucrose gradients it had a density of 1.17 g/cm3 and sedimented with the same s value as Bunyamwera virus. The diameter of negatively stained virions was approximately 110 nm. Three species of RNA sedimenting at 30S (L), 26S (M) and 14S (S) and four virion proteins (L, G1, G2, N) were detected in preparations of purified virions. The results were consistent with the classification of Leanyer virus as a member of the family Bunyaviridae, possibly within the Bunyavirus genus.

The Mapputta group of arboviruses: ultrastructural and molecular studies which place the group in the bunyavirus genus of the family Bunyaviridae.

We have characterized members of the Mapputta group of 'bunyavirus-like' viruses in terms of morphology, structure, ultrastructural development and virus-directed RNA and protein synthesis. Our primary study has been with Maprik virus (MPK) as a representative of the group. The MPK virion is uniformly spherical (congruent to 90 nm diameter) and possesses a membrane envelope. Virus maturation is by budding into small vesicles in the perinuclear region. During infection of BHK cells which is cytopathic, maximum viral RNA synthesis is congruent to 20% of RNA synthesis in uninfected cells without actinomycin D. In Aedes albopictus cells, where high initial titres are followed by a persistent infection, maximum viral RNA synthesis is only 1.5% of levels in control cells. MPK virions contain three RNA species. MPK generates four virus-specific RNAs (L, M, S1 and S2) and three nucleocapsid species in infected BHK cells. Six virus-specific polypeptides are present in infected cells; the four largest correspond to L (a probable transcriptase component), G1 and G2 (envelope proteins) and N (the nucleocapsid protein), respectively. These results and complementary studies with Trubanaman and Gan Gan viruses indicate that the members of the Mapputta serogroup can be placed in the bunyavirus genus of the family Bunyaviridae.

Structural proteins of Marituba virus (Bunyaviridae).

Marituba virus was purified by rate-zonal sedimentation and equilibrium density centrifugation in sucrose gradients. The buoyant density of virus particles was 1.19 g/ml. Purified virus was dissociated and its proteins were analyzed by SDS-PAGE. Four virus polypeptides were identified and designated L, G1, G2 and N. Their average molecular weights were 190 x 10(3) [L, range (180-200) x 10(3)], 120 x 10(3) [G1, range (118-122) x 10(3)], 26 x 10(3) [G2, range (24-28) x 10(3)], and 20 x 10(3) [N, range (19-21) x 10(3)]. Polypeptides N and L are, respectively, the major and the minor viral components. G1 and G2 are glycopeptides as demonstrated by the preferential incorporation of labeled 3H-glucosamine.

Characterization of the oligosaccharides of Inkoo virus envelope glycoproteins.

Inkoo virus (a bunyavirus) was grown in BHK-21 cells and labelled with [35S]methionine or [3H]mannose. [35S]Methionine labelled the two envelope glycoproteins G1 (Mr = 125000) and G2 (Mr = 35000), as well as the nucleocapsid protein N (Mr = 25000). Only G1 and G2 were labelled with the sugar precursor. The [3H]mannose-labelled virus was solubilized with detergent and digested with Pronase. The structure of the labelled glycopeptides originating from the mixture of G1 and G2 was studied by degrading the glycans stepwise with specific exo- and endoglycosidases, and by analysing the products by both gel and paper chromatography, as well as lectin-affinity chromatography. Three classes of N-glycosidic glycans were found: complex glycans with the monosaccharide sequence (NeuNAc alpha Gal beta GlcNac beta) greater than or equal to 2 (Man)3 (GlcNAc)2 (occurrence of fucose was not studied), high mannose-type chains with the average structure (Man)4-6 (GlcNAc)2, and endoglycosidase H-resistant small glycans which were partly susceptible to mannosidase. These latter types of oligosaccharide chains are a novel finding among virus glycoproteins. The relative ratio of the three types of oligosaccharide chains was roughly 4 . 6:1:1 respectively. The G1 glycoprotein carried most of the sugar chains, since it contained 85% of the [3H]mannose label. The results are discussed in relation to the site of virus maturation at smooth-surfaced vesicles in the Golgi region.

Seven infection-specific polypeptides in BHK cells infected with Bunyamwera virus.

Virus-specific polypeptide synthesis was examined in BHK cells and Vero cells infected with Bunyamwera virus. In BHK cells, in addition to the four previously reported virus-coded proteins (L, G1, G2, and N), three other infection-specific proteins were detected. These proteins, of nominal molecular weight 50,000 (p50), 16,000 (p16), and 13,000 (p13), were not labeled in mock-infected cells, were first synthesized between 4 and 8 h after infection, and were relatively prominent among the limited number of proteins generated late in infection. In preparations of purified Bunyamwera virus from BHK cell supernatants, p16 was detected but not p50 or p13. In Vero cells infected with Bunyamwera virus, both p50 and p13 were labeled strongly. Maprik virus, a member of the Mapputta group of arboviruses, is a member of the Bunyavirus genus (S.E. Newton, unpublished data). Maprik virus did not induce the synthesis of p50, p16, or p13; however, two smaller proteins (p17 and p15) which may correspond to p16 and p13 were labeled late in Maprik infection. Our data argue that p16 is a virus-coded component of the Bunyamwera virus particle and that p50 and p13 are virus-coded, nonstructural proteins.

The 3' terminal RNA sequences of bunyaviruses and nairoviruses (Bunyaviridae): evidence of end sequence generic differences within the virus family.

The 3' terminal nucleotide sequences of the three virus RNA species of viruses representing eight serogroups of bunyaviruses (genus Bunyavirus, Bunyaviridae) and six serogroups of nairoviruses (genus Nairovirus, Bunyaviridae) have been characterized. Members of the Bunyavirus genus have conserved 3' end sequences (generally, 3' UCAUCACAUGA...) that differ from the conserved 3' end sequences of members of the Nairovirus genus (generally, 3' AGAGUUUCU...).

Identification of a major non-structural protein in the nuclei of Rift Valley fever virus-infected cells.

A non-structural protein of mol. wt. 34 X 10(3) was demonstrated in the nuclei of Rift Valley fever virus-infected Vero cells by SDS-polyacrylamide gel electro-phoresis. The protein appears to correspond to the virus-induced antigen demonstrated by indirect immunofluorescence in intranuclear inclusions.

Monosaccharide sequence of protein-bound glycans of Uukuniemi virus.

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [(3)H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [(3)H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)(3)(GlcNAc)(2) peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [(3)H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [(35)S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.