Structural proteins of La Crosse virus.

Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm).     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Electron microscopy of vitrified-hydrated La Crosse virus.

La Crosse (LAC) virions were cryopreserved by rapid freezing in a thin layer of vitreous ice. The vitrified-hydrated LAC virions were subsequently imaged at -170 degrees C in a transmission electron microscope equipped with a low-temperature specimen holder. This cryoelectron microscopic technique eliminates the artifacts frequently associated with negative staining. Images of vitrified-hydrated LAC virions clearly revealed surface spikes as well as bilayer structure. Size measurements of the vitrified-hydrated LAC virions showed heterogeneity, with diameters ranging from 75 to 115 nm. Regardless of the particle size, the spike was about 10 nm long, and the bilayer was about 4 nm thick. The spikes are interpreted to be one or both of the glycoproteins, and the bilayer is interpreted to be the membrane envelope of the virus. In contrast to the pleomorphic appearance of the negatively stained LAC virions, the vitrified-hydrated LAC virions showed uniform spherical shapes regardless of their sizes.     

The ends of La Crosse virus genome and antigenome RNAs within nucleocapsids are base paired.

The three La Crosse virus genomes are found as circular structures in the electron microscope, and the RNA ends of at least the small (S) and medium (M) segments are highly complementary. When examined for psoralen cross-linking, about half of the S, at most 1 to 2% of the M, and none of the large (L) nucleocapsid RNAs could be cross-linked in virions or at late times intracellularly, under conditions in which each free RNA reacted completely. For the S segment, genomes and antigenomes first detected intracellularly could not be cross-linked at all, and their cross-linkability increased gradually with time. Antigenomes behaved similarly to genomes in all respects. It appears that the majority of all three segments are base paired at their ends and that the limited cross-linkability reflects the accessability of the RNA within nucleocapsids to psoralen. The gradual increase in cross-linkability may be important in persistent mosquito cell infection, in which it correlates with decreased S mRNA synthesis rates, and may be part of the mechanism which this infection becomes self-limiting. The implications of double-stranded RNA panhandles within nucleocapsids are discussed.     

Segmented genome and nucleocapsid of La Crosse virus.

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Virulence of La Crosse virus is under polygenic control.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.     

La Crosse virus infection of mammalian cells induces mRNA instability.

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.     

Nucleotide sequences of three poriferan 5 S ribosomal RNAs.

We have determined the nucleotide sequences of 5 S ribosomal RNAs isolated from the sponges Halichondria panicea, Hymeniacidon sanguinea, and Haliclona oculata. The structures can be fitted in a universal five-helix secondary structure model (De Wachter, Chen and Vandenberghe (1982) Biochimie 64, 311-329) applicable to all 5 S RNAs hitherto sequenced. The base pairing scheme proves to be extremely conserved throughout the metazoan kingdom, yet four slightly different variants of the model may be distinguished among the 5 S RNAs from the seven animal phyla investigated until now.     

Nucleotide sequence analysis of the long terminal repeat of avian myeloblastosis virus and adjacent host sequences.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.     

Anti-mRNAs in La Crosse bunyavirus-infected cells.

Unlike some members of the family Bunyaviridae which contain ambisense genomes, all La Crosse virus reading frames are translated from antigenome sense mRNAs. Nevertheless, La Crosse virus genome sense mRNAs or anti-mRNAs are initiated from antigenome templates. These are characterized by the same range of capped, nontemplated sequences at their 5' ends as mRNAs, but their 3' ends are presumed to be heterogenous, as they were not seen on RNA blots. The anti-mRNAs are estimated to be 15 to 30 times less abundant than mRNAs, but remarkably, this ratio is similar to that of functional genome sense mRNAs made from other bona fide ambisense segments. A role for these anti-mRNAs during infection is unclear.     

Comparison of the sequences and coding of La Crosse and snowshoe hare bunyavirus S RNA species.

The sequence of the S RNA of La Crosse bunyavirus was deduced from analyses of DNA copies cloned in the Escherichia coli plasmid pBR322. The S RNA is 984 nucleotides in length, has a base ratio of 31.8% U, 27.0% A, 23.2% C, and 18.0% G, and codes for two distinct gene products that are read from overlapping reading frames in the viral complementary strand. The larger gene product (N, 26.5 x 10(3) daltons) contains 235 amino acids, and the smaller gene product (NSS, 10.4 x 10(3) daltons) has 92 amino acids. Comparisons with the published sequences of the related snowshoe hare bunyavirus S RNA and its gene products (Bishop et al., Nucleic Acids Res. 10:3703-3713, 1982) indicate that there are a total of 114 nucleotide differences (6 additions or deletions and 108 substitutions). Also, there are 22 amino acid differences between the N proteins and 12 amino acid differences between the NSS proteins of the two S RNAs.     

La Crosse virus production and export have a colchicine-sensitive step

In the present investigation, the effect of colchicine on La Crosse virus production and export was tested. Colchicine-treated, La Crosse virus-infected cells: (1) had decreased mean virus titers compared with those of control cells; (2) had a ratio of released to cell-associated virus of 1–1.9 whereas control cells had a ratio of 13. A colchicine-sensitive step, possibly involving microtubules, may be involved in virus production and/or release from the cell.     

La Crosse virus: replication in vertebrate and invertebrate hosts

La Crosse virus is maintained in a cycle involving mosquitoes and small mammals. Vertebrate cell infection is generally cytolytic; vector cell infection results in persistent infection. Features of La Crosse virus replication that may permit the virus to traffic between vector and vertebrate hosts and condition different infection outcomes are described.