Analysis of the medium (M) segment sequence of Guaroa virus and its comparison to other orthobunyaviruses

Thomas Briese1, Andrew Rambaut2 and W. Ian Lipkin1

1 Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York, NY 10032, USA
2 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Correspondence
Thomas Briese
thomas.briese{at}columbia.edu


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Guaroa virus (GROV), a segmented virus in the genus Orthobunyavirus, has been linked to the Bunyamwera serogroup (BUN) through cross-reactivity in complement fixation assays of S segment-encoded nucleocapsid protein determinants, and also to the California serogroup (CAL) through cross-reactivity in neutralization assays of M segment-encoded glycoprotein determinants. Phylogenetic analysis of the S-segment sequence supported a closer relationship to the BUN serogroup for this segment and it was hypothesized that the serological reaction may indicate genome-segment reassortment. Here, cloning and sequencing of the GROV M segment are reported. Sequence analysis indicates an organization similar to that of other orthobunyaviruses, with genes in the order GN–nsm–gC, and mature proteins generated by protease cleavage at one, and by signalase at possibly three, sites. A potential role of motifs that are more similar to CAL than to BUN virus sequences with respect to the serological reaction is discussed. No discernable evidence for reassortment was identified.

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY380581.


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Like other orthobunyaviruses, Guaroa virus (GROV) has a segmented, negative-strand RNA genome that is comprised of three segments, named small (S), medium (M) and large (L) (Bouloy et al., 1973; Gentsch & Bishop, 1976; Clewley et al., 1977; El Said et al., 1979). The S segment of orthobunyaviruses encodes the nucleocapsid protein (N) and a non-structural protein (NSs) that may modulate viral polymerase activity, and acts as an alpha/beta interferon antagonist (Gentsch & Bishop, 1978; Bouloy et al., 1984; Elliott, 1985; Bridgen et al., 2001; Weber et al., 2001). The genome-complementary strand of the M segment includes one open reading frame (ORF) for a polyprotein that yields the two surface glycoproteins GN and gC (G2 and G1, respectively; Lappin et al., 1994) and a non-structural protein (NSm) of unknown function (Gentsch & Bishop, 1979; Fuller & Bishop, 1982; Elliott, 1985; Fazakerley et al., 1988; Nakitare & Elliott, 1993). The L segment directs expression of a large, virion-associated protein with RNA-dependent RNA polymerase activity (Bouloy & Hannoun, 1976; Obijeski et al., 1976; Elliott, 1989; Endres et al., 1989; Jin & Elliott, 1991).

The International Committee on Taxonomy of Viruses considers Guaroa virus to be a species distinct from the species California encephalitis virus (CEV) and Bunyamwera virus (BUNV) within the genus Orthobunyavirus of the family Bunyaviridae (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm); some investigators have suggested that GROV should not be included in either the California serogroup (CAL) or the Bunyamwera serogroup (BUN) (Whitman & Shope, 1962; Calisher & Maness, 1970; Wellings et al., 1971; Hunt & Calisher, 1979; Klimas et al., 1981). Serological assays have shown some link of GROV to both serogroups. In complement fixation (CF) assays, serological cross-reactivity was observed with BUN, but not CAL, members. In contrast, in haemagglutination–inhibition (HI) and neutralization (NT) assays, cross-reactivity was evident with CAL, but not BUN, members (Groot et al., 1959; Casals & Whitman, 1960; Whitman & Shope, 1962; Tauraso, 1969). Results similar to those of CF assays were obtained in immunodiffusion, showing no cross-reactivity between GROV and CAL viruses, but weak cross-reaction with BUNV and Tensaw virus (Calisher & Maness, 1970; Wellings et al., 1970). Immunoelectrophoresis, however, indicated common determinants between GROV and CAL viruses (Wellings et al., 1971). Reaction in CF assays is determined by N, whereas reaction in NT/HI assays is determined by the glycoproteins (Lindsey et al., 1977; Gentsch et al., 1980; González-Scarano et al., 1982; Kingsford & Hill, 1983; Ludwig et al., 1991). Discordant serological reaction may therefore indicate different phylogenetic relationships for GROV N (S segment) and the glycoproteins (M segment). S-segment sequencing suggested a closer relationship to BUN than to CAL viruses; it has thus been hypothesized that GROV may be a reassortant virus (Dunn et al., 1994). Here, we report the GROV M-segment sequence and its analysis in comparison to other M-segment sequences.

GROV RNA was reverse-transcribed by using Superscript II (Invitrogen) and amplified by PCR (Saiki et al., 1985) using primers (1·6 µM; Table 1), dNTPs (200 µM), MgCl2 (Table 1) and BIO-X-ACT polymerase (Bioline) in a PTC-200 thermocycler (MJ Research) for 45 cycles of 1 min at 92 °C, 1 min at 45–53 °C and 1–2·5 min at 68 °C (Table 1). Products were cloned and sequenced (Sanger et al., 1977); analysis using the Wisconsin GCG package (Accelrys) indicated one ORF of 4254 nt (1418 aa) for the assembled sequence (GenBank accession no. AY380581).


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Table 1. Amplification primers

 
Downstream of an untranslated region, the antigenomic strand encodes a protein that is related to GN of other BUN and CAL viruses (nt 20–943; 35 kDa). The N-terminal sequence is consistent with a functional signal peptide for membrane translocation (Blobel & Dobberstein, 1975; Lingappa et al., 1978; von Heijne, 1988), similar to other viruses of the genus (Fazakerley et al., 1988). In contrast to other orthobunyavirus M-segment sequences, GROV contains three potential AUG codons, with the first one being in the best context according to Kozak's rules (–3=A, +4=G; Kozak, 1986, 1991). This potentially results in a N-terminally extended product (Fig. 1). Cleavage of the signal peptide at T21 with respect to the first methionine is compatible with conservation of terminal tripeptides, as suggested by Lees et al. (1986), although SPV would change to TPV and RCF to KCF. Prediction of signalase cleavage at T21 or P23 by SignalP-NN/–HMM (http://www.cbs.dtu.dk/services/; Nielsen et al., 1997) supports this view (data not shown).




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Fig. 1. Alignment of M-segment sequences of selected CAL [Jamestown Canyon virus (JCV), MELV, Keystone virus (KEYV), Lumbo virus (LUMV), LACV, CEV and TVTV] and BUN (GERV, CVV and BUNV) viruses with that of GROV. ___, Potential transmembrane regions; h, potential h-region of predicted signal sequence; |, potential proteolytic cleavage; *, conserved cysteine; ///, epitopes identified by Cheng et al. (2000); +++, potential glycosylation site; v, conservation between GROV and CAL serogroup virus sequences; xxx, conserved cleavage motif at C-terminus of GN; bold letters indicate predicted n-terminus of gC; ttt, potential trypsin cleavage site; #, amino acid position involved in neutralizing epitope of LACV (Bupp & González-Scarano, 1998). Amino acid positions for the GROV sequence are indicated at the end of each line.

 
The predicted amino acid sequence for GN contains the sequence KSLRV/AAR, allowing protease cleavage to separate mature GN from the downstream NSm analogue (xxxx|; Fig. 1) (Fazakerley et al., 1988). The NSm-like sequence is characterized by a conserved, N-terminal, hydrophobic sequence followed by a short deletion, when compared to other M-segment sequences, and a motif that is conserved amongst BUN and CAL viruses (G416DFc/t/sNKCg/rf/qC425). Little conservation was observed around the NSm/GC junction, so a potential site for cleavage, possibly executed by signalase (Fazakerley et al., 1988), is not apparent. Cleavage after a conserved alanine residue (A475), analogous to the termination of NSm in CAL viruses (Campbell & Huang, 1999), is possible. This would result in nt 944–1444 encoding NSm (19 kDa). Analysis of the junction by SignalP predicts three cleavage sites with similar likelihoods: A472, A475 and A479 (data not shown). Cleavage after A472 would result in positions –3=V and –1=A, one of the most frequent combinations in signalase sites. Cleavage after A479 would imply a long c-region, but would result in an N-terminal GC-tripeptide E480EP, similar to BUNV and Cache Valley virus (CVV) [Fig. 1; Germiston virus (GERV) N-terminus deduced from alignments (Lees et al., 1986; Gerbaud et al., 1992; Lappin et al., 1994); SignalP prediction, ATM-LV or VVA-GE]. Cleavage after any of the three alanines in GROV occurs close to a potential glycosylation site (N492), but even cleavage at A479 would be at the ‘minimum glycosylation distance’ of 13 aa that has been determined for cleaved internal signals (Nilsson et al., 1994).

The N-terminal portion of the next protein (nt 1445–4273; 108 kDa) is surprisingly divergent from other GC proteins. The C-terminal moiety, beginning about 150 aa after a conserved potential trypsin cleavage site (tttt, Fig. 1; Fazakerley et al., 1988), is again conserved when compared to other M-segment sequences. The 3' non-coding region shows little conservation and is longer than those of the other M segments.

Five potential glycosylation sites were identified (+++; Fig. 1), including an N-terminal site in GC that is conserved among gC sequences of the cal viruses and is found in approximately the same position in grov (n492); an n-terminal glycosylation site that is conserved in gC sequences of sequenced BUN viruses was not found in the majority of GROV clones analysed. Among 10 clones, six carried AATGACAtA for N616DI (---; Fig. 1), whereas four carried AATGACAcA, encoding the potential glycosylation site N616DT.

Analysis by TopPred2 (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html; Claros & von Heijne, 1994) predicts six major transmembrane regions. Two are predicted for GN: the signal peptide (aa 6–26) and a second region that is compatible with a stop-transfer signal/membrane anchor (aa 210–230), which would result in a cytoplasmic location for a strictly conserved downstream KTY motif, a stretch of mostly hydrophobic residues that includes two proline residues, and the protease cleavage motif. Three transmembrane regions are predicted for NSm (aa 315–335, 368–388 and 455–475); the first, located 10 aa downstream of the GN C-terminus, is compatible with a third internal signal sequence, with cleavage predicted after G337 by SignalP. Signalase cleavage close to a cytoplasmic protease site may resemble the proposed situation at the C/prM cleavage site of flaviviruses (Stocks & Lobigs, 1998; Amberg & Rice, 1999). One transmembrane region (aa 1372–1392), a potential membrane anchor (Fazakerley et al., 1988; Pekosz et al., 1995), is predicted for GC. The overall topology appears to be well-conserved, as indicated by conservation of the same cysteine residues as in the polyproteins of all other BUN and CAL viruses (Fig. 1) (Lees et al., 1986; Grady et al., 1987; Pardigon et al., 1988). Conservation of sequence motifs with respect to CAL but not BUN virus sequences is noted for K149, Q163 and P299 in GN, P347 and N455/F456 in NSm, and H591QH, G601EKCNSA607, E958, K1036, G1243 and K1411/K1412 in GC.

Pairwise, sliding-window distance analysis (SimPlot; http://sray.med.som.jhmi.edu/RaySoft/SimPlot/; Lole et al., 1999) between GROV and BUN and CAL viruses indicated an almost equidistant position of GROV, with lowest distance scores in the GN region (approx. position 1–300; Fig. 2a) and highest scores obtained in the NSm sequence (approx. position 300–500) and in the N-terminal portion of GC (approx. position 500–1400). Serogroup-specific differences appear to be most pronounced in three regions (approx. positions 100–200, 550–650 and 1200–1275), where a separation between sequences of BUNV, CVV and GERV and CEV, Melao virus (MELV) and Trivittatus virus (TVTV) is observed. GROV appears to be less distant from BUNV/GERV/CVV than from CEV/MELV/TVTV in all regions except for the second part of the second region (at the N-terminus of GC toward N616), where CEV/MELV/TVTV are less distant from GROV than BUNV/GERV; however, CVV remains the most closely related sequence throughout. In a reconstructed phylogenetic tree, the GROV M-segment sequence is placed in a closer relationship to sequences of BUN than of CAL viruses (62 % bootstrap support; Fig. 2b). Phylogenetic relationships of each individual ORF are similar to that of the entire sequence (data not shown).



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Fig. 2. Phylogenetic analysis of the GROV M-segment sequence. (a) Sliding-window distance analysis between GROV, BUN viruses BUNV, GERV and CVV and CAL viruses CEV, MELV and TVTV (amino acid sequence; window, 60 aa, step, 10 aa). (b) Reconstructed phylogenetic maximum-likelihood tree for nucleotide sequence. The tree was constructed by using the ‘subtree prune regraft’ (SPR) heuristic-search strategy under the general time-reversible model of nucleotide substitution with site-specific rate heterogeneity, modelled by using the discrete gamma distribution (Yang, 1994). Parameters were initially estimated on a neighbour-joining tree. Bootstrap support resulting from 500 SPR heuristic-search replicates is indicated for relevant branches. Oropouche virus (Simbu serogroup) served as an outgroup to root the tree. GenBank accession numbers are shown in parentheses. Abbreviations: INKV, Inkoo virus; JSV, Jerry Slough virus; SAV, San Angelo virus; SDNV, Serra do Navio virus; SORV, South River virus.

 
A genetic distance of GROV from both serogroups is compatible with biological features. Genetic interference has been observed between CAL viruses in experiments that mimic interrupted feeding behaviour of mosquitoes (Beaty et al., 1985; Sundin & Beaty, 1988). Interference was not observed between CAL viruses and GROV (Beaty et al., 1983). Cell-culture experiments, however, indicated genetic distance not only from CAL, but also from BUN, viruses. Whilst genome-segment reassortment between GROV mutants was observed, heterologous reassortment was not observed between GROV and La Crosse virus (LACV), snowshoe hare virus (SSHV), TVTV or Tahyna virus (TAHV) (Gentsch et al., 1980), but also not between GROV and BUNV, Maguari virus or Batai virus (Iroegbu & Pringle, 1981).

The structural determinants of GROV's unique serological reaction pattern are obscure. GN is not a major target of neutralizing antibodies that interfere with infection of mammalian cells (Ludwig et al., 1989; Cheng et al., 2000) and the few amino acids that are conserved in GN between grov and cal viruses do not correlate with identified epitopes of gN (/; Fig. 1; Cheng et al., 2000). Therefore, GN is unlikely to form major determinants of the reaction of GROV in NT/HI assays. Likewise, NSm is unlikely to be involved. Epitopes detected in NT/HI assays have been mapped to the N-terminal portion of GC, mainly in relation to the trypsin site of LACV/SSHV (González-Scarano et al., 1982; Kingsford et al., 1983; Najjar et al., 1985; Kingsford & Boucquey, 1990). However, their relation to primary amino acid sequence is not defined and only in one case has a particular amino acid that is involved in neutralization been identified (residue 29 of LACV GC; #, Fig. 1; Bupp & González-Scarano, 1998). The divergence of this region in comparison to available BUN virus sequences may explain the lack of cross-reaction between GROV and these viruses in NT/HI assays. Although also divergent when compared to sequences of CAL viruses, this region does contain motifs that are conserved with respect to CAL, but not BUN, viruses (H591QH and G601EKCNSA607) and two glycosylation sites. N492, which is conserved amongst CAL but not BUN virus sequences, is present, whereas N616, which is conserved amongst BUN but not CAL virus sequences, is only present in a minority of GROV clones. N492 flanks the first putative antigenic domain that was proposed by Brockus & Grimstad (2001) and both conserved amino acid motifs and N616 are located in their second putative antigenic domain. It is conceivable that these positions contribute to the serological reaction of GROV. The mutation at the glycosylation site N616DI/T is intriguing, given that M-segment sequence has been associated with plaque size (Iroegbu & Pringle, 1981) and the observation that GROV can generate both large and small plaque morphologies, of which only the small variant elicited antibodies that were cross-reactive with CEV and TAHV (Tauraso, 1969).

In summary, our analysis of the GROV M-segment sequence indicates a relative phylogenetic relationship that is comparable to that reported for the GROV S-segment sequence (Fig. 2b; Dunn et al., 1994), and does not provide evidence for genome-segment reassortment. Instead, in a sequence that is almost equidistant to published CAL and BUN virus sequences, isolated determinants in the N-terminal portion of GC were identified that potentially relate to the unique serological reaction pattern of GROV and are more compatible with GROV forming a bridge between both serogroups, as originally proposed by Whitman & Shope (1962).


   ACKNOWLEDGEMENTS
 
We thank Bob Tesh for supplying the Guaroa virus stock, Wuxia Fu for excellent technical assistance and Charles Calisher for many helpful suggestions and discussions. This work was supported by awards from the Ellison Medical Foundation and NIH (AI 056118) to T. B. and W. I. L.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Amberg, S. M. & Rice, C. M. (1999). Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 73, 8083–8094.[Abstract/Free Full Text]

Beaty, B. J., Bishop, D. H. L., Gay, M. & Fuller, F. (1983). Interference between bunyaviruses in Aedes triseriatus mosquitoes. Virology 127, 83–90.[CrossRef][Medline]

Beaty, B. J., Sundin, D. R., Chandler, L. J. & Bishop, D. H. L. (1985). Evolution of bunyaviruses by genome reassortment in dually infected mosquitoes (Aedes triseriatus). Science 230, 548–550.[Medline]

Blobel, G. & Dobberstein, B. (1975). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67, 835–851.[Abstract]

Bouloy, M. & Hannoun, C. (1976). Studies on Lumbo virus replication. I. RNA-dependent RNA polymerase associated with virions. Virology 69, 258–264.[CrossRef][Medline]

Bouloy, M., Krams-Ozden, S., Horodniceanu, F. & Hannoun, C. (1973). Three-segment RNA genome of Lumbo virus (Bunyavirus). Intervirology 2, 173–180.[Medline]

Bouloy, M., Vialat, P., Girard, M. & Pardigon, N. (1984). A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein. J Virol 49, 717–723.[Medline]

Bridgen, A., Weber, F., Fazakerley, J. K. & Elliott, R. M. (2001). Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc Natl Acad Sci U S A 98, 664–669.[Abstract/Free Full Text]

Brockus, C. L. & Grimstad, P. R. (2001). Comparative analysis of G1 glycoprotein-coding sequences of Cache Valley virus (Bunyaviridae: Bunyavirus) isolates. Virus Genes 22, 133–139.[CrossRef][Medline]

Bupp, K. & González-Scarano, F. (1998). Pseudotype formation with La Crosse virus glycoproteins. J Gen Virol 79, 667–671.[Abstract]

Calisher, C. H. & Maness, K. S. C. (1970). Arbovirus identification by an agar-gel diffusion technique. Appl Microbiol 19, 557–564.[Medline]

Campbell, W. P. & Huang, C. (1999). Sequence comparisons of medium RNA segment among 15 California serogroup viruses. Virus Res 61, 137–144.[CrossRef][Medline]

Casals, J. & Whitman, L. (1960). A new antigenic group of arthropod-borne viruses: the Bunyamwera group. Am J Trop Med Hyg 9, 73–77.[Medline]

Cheng, L. L., Schultz, K. T., Yuill, T. M. & Israel, B. A. (2000). Identification and localization of conserved antigenic epitopes on the G2 proteins of California serogroup bunyaviruses. Viral Immunol 13, 201–213.[Medline]

Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685–686.[Medline]

Clewley, J., Gentsch, J. & Bishop, D. H. L. (1977). Three unique viral RNA species of snowshoe hare and La Crosse bunyaviruses. J Virol 22, 459–468.[Medline]

Dunn, E. F., Pritlove, D. C. & Elliott, R. M. (1994). The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis. J Gen Virol 75, 597–608.[Abstract]

Elliott, R. M. (1985). Identification of nonstructural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae). Virology 143, 119–126.[Medline]

Elliott, R. M. (1989). Nucleotide sequence analysis of the small (S) RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae. J Gen Virol 70, 1281–1285.[Abstract]

El Said, L. H., Vorndam, V., Gentsch, J. R. & 7 other authors (1979). A comparison of La Crosse virus isolates obtained from different ecological niches and an analysis of the structural components of California encephalitis serogroup viruses and other bunyaviruses. Am J Trop Med Hyg 28, 364–386.[Medline]

Endres, M. J., Jacoby, D. R., Janssen, R. S., González-Scarano, F. & Nathanson, N. (1989). The large viral RNA segment of California serogroup bunyaviruses encodes the large viral protein. J Gen Virol 70, 223–228.[Abstract]

Fazakerley, J. K., González-Scarano, F., Strickler, J., Dietzschold, B., Karush, F. & Nathanson, N. (1988). Organization of the middle RNA segment of snowshoe hare bunyavirus. Virology 167, 422–432.[CrossRef][Medline]

Fuller, F. & Bishop, D. H. L. (1982). Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells. J Virol 41, 643–648.[Medline]

Gentsch, J. & Bishop, D. H. L. (1976). Recombination and complementation between temperature-sensitive mutants of a bunyavirus, snowshoe hare virus. J Virol 20, 351–354.[Medline]

Gentsch, J. R. & Bishop, D. H. L. (1978). Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein. J Virol 28, 417–419.[Medline]

Gentsch, J. R. & Bishop, D. H. L. (1979). M viral RNA segment of bunyaviruses codes for two glycoproteins, G1 and G2. J Virol 30, 767–770.[Medline]

Gentsch, J. R., Rozhon, E. J., Klimas, R. A., El Said, L. H., Shope, R. E. & Bishop, D. H. (1980). Evidence from recombinant bunyavirus studies that the M RNA gene products elicit neutralizing antibodies. Virology 102, 190–204.[CrossRef][Medline]

Gerbaud, S., Pardigon, N., Vialat, P. & Bouloy, M. (1992). Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins. J Gen Virol 73, 2245–2254.[Abstract]

González-Scarano, F., Shope, R. E., Calisher, C. E. & Nathanson, N. (1982). Characterization of monoclonal antibodies against the G1 and N proteins of LaCrosse and Tahyna, two California serogroup bunyaviruses. Virology 120, 42–53.[CrossRef][Medline]

Grady, L. J., Sanders, M. L. & Campbell, W. P. (1987). The sequence of the M RNA of an isolate of La Crosse virus. J Gen Virol 68, 3057–3071.[Abstract]

Groot, H., Oya, A., Bernal, C. & Barreto-Reyes, P. (1959). Guaroa virus, a new agent isolated in Colombia, South America. Am J Trop Med Hyg 8, 604–609.[Medline]

Hunt, A. R. & Calisher, C. H. (1979). Relationships of bunyamwera group viruses by neutralization. Am J Trop Med Hyg 28, 740–749.[Medline]

Iroegbu, C. U. & Pringle, C. R. (1981). Genetic interactions among viruses of the Bunyamwera complex. J Virol 37, 383–394.[Medline]

Jin, H. & Elliott, R. M. (1991). Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses. J Virol 65, 4182–4189.[Medline]

Kingsford, L. & Hill, D. W. (1983). The effect of proteolytic cleavage of La Crosse virus G1 glycoprotein on antibody neutralization. J Gen Virol 64, 2147–2156.[Abstract]

Kingsford, L. & Boucquey, K. H. (1990). Monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus that react with other California serogroup viruses. J Gen Virol 71, 523–530.[Abstract]

Kingsford, L., Ishizawa, L. D. & Hill, D. W. (1983). Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the G1 glycoprotein of La Crosse virus. Virology 129, 443–455.[CrossRef][Medline]

Klimas, R. A., Ushijima, U., Clerx-van Haaster, C. M. & Bishop, D. H. L. (1981). Radioimmune assays and molecular studies that place Anopheles B and Turlock serogroup viruses in the Bunyavirus genus (Bunyaviridae). Am J Trop Med Hyg 30, 876–887.[Medline]

Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292.[Medline]

Kozak, M. (1991). Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266, 19867–19870.[Free Full Text]

Lappin, D. F., Nakitare, G. W., Palfreyman, J. W. & Elliott, R. M. (1994). Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm. J Gen Virol 75, 3441–3451.[Abstract]

Lees, J. F., Pringle, C. R. & Elliott, R. M. (1986). Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product. Virology 148, 1–14.[CrossRef][Medline]

Lindsey, H. S., Klimas, R. A. & Obijeski, J. F. (1977). La Crosse virus soluble cell culture antigen. J Clin Microbiol 6, 618–626.[Medline]

Lingappa, V. R., Katz, F. N., Lodish, H. F. & Blobel, G. (1978). A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function. J Biol Chem 253, 8667–8670.[Abstract]

Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73, 152–160.[Abstract/Free Full Text]

Ludwig, G. V., Christensen, B. M., Yuill, T. M. & Schultz, K. T. (1989). Enzyme processing of La Crosse virus glycoprotein G1: a bunyavirus-vector infection model. Virology 171, 108–113.[CrossRef][Medline]

Ludwig, G. V., Israel, B. A., Christensen, B. M., Yuill, T. M. & Schultz, K. T. (1991). Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus. Microb Pathog 11, 411–421.[CrossRef][Medline]

Najjar, J. A., Gentsch, J. R., Nathanson, N. & González-Scarano, F. (1985). Epitopes of the G1 glycoprotein of La Crosse virus form overlapping clusters within a single antigenic site. Virology 144, 426–432.[CrossRef][Medline]

Nakitare, G. W. & Elliott, R. M. (1993). Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm. Virology 195, 511–520.[CrossRef][Medline]

Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1–6.[CrossRef]

Nilsson, I., Whitley, P. & von Heijne, G. (1994). The COOH-terminal ends of internal signal and signal–anchor sequences are positioned differently in the ER translocase. J Cell Biol 126, 1127–1132.[Abstract]

Obijeski, J. F., Bishop, D. H. L., Murphy, F. A. & Palmer, E. L. (1976). Structural proteins of La Crosse virus. J Virol 19, 985–997.[Medline]

Pardigon, N., Vialat, P., Gerbaud, S., Girard, M. & Bouloy, M. (1988). Nucleotide sequence of the M segment of Germiston virus: comparison of the M gene product of several bunyaviruses. Virus Res 11, 73–85.[Medline]

Pekosz, A., Griot, C., Stillmock, K., Nathanson, N. & González-Scarano, F. (1995). Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies. J Virol 69, 3475–3481.[Abstract]

Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.[Medline]

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.[Abstract]

Stocks, C. E. & Lobigs, M. (1998). Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. J Virol 72, 2141–2149.[Abstract/Free Full Text]

Sundin, D. R. & Beaty, B. J. (1988). Interference to oral superinfection of Aedes triseriatus infected with La Crosse virus. Am J Trop Med Hyg 38, 428–432.[Medline]

Tauraso, N. M. (1969). Identification of two plaque variants of Guaroa virus. Arch Gesamte Virusforsch 28, 212–218.[Medline]

von Heijne, G. (1988). Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta 947, 307–333.[Medline]

Weber, F., Dunn, E. F., Bridgen, A. & Elliott, R. M. (2001). The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system. Virology 281, 67–74.[CrossRef][Medline]

Wellings, F. M., Sather, G. E. & Hammon, W. M. (1970). A type-specific immunodiffusion technique for the California encephalitis virus group. J Immunol 105, 1194–1200.[Medline]

Wellings, F. M., Sather, G. E. & Hammon, W. M. (1971). Immunoelectrophoretic studies of the California encephalitis virus group. J Immunol 107, 252–259.[Medline]

Whitman, L. & Shope, R. (1962). The California complex of arthropod-borne viruses and its relationship to the Bunyamwera group through Guaroa virus. Am J Trop Med Hyg 11, 691–696.[Medline]

Yang, Z. (1994). Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J Mol Evol 39, 306–314.[Medline]

Received 20 March 2004; accepted 21 June 2004.