Organization of the M genomic segment of Toscana phlebovirus.

Abstract

The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3' NSm-G1 -G2 5'

Full text

Page 1

J ournal of General Virology (1997), 78, 77–81.Printed in Great Britain
............................................................................................................................................................................................................... SHORT COMMUNICATION
Organization of the M genomic segment of Toscana
phlebovirus
P. Di Bonito, S. Mochi, M. C. Gro ? , D. Fortini and C. Giorgi
Laboratory of Virology, Istituto Superiore di Sanita ? , Viale Regina Elena 299, 00161 Rome, Italy
The nucleotide sequence of the Toscana (TOS) virus
M RNA segment contains a single major open
reading frame in the viral-complementary sequ-
ence, which can encode a polyprotein of 1339
aminoacids.TomaptheTOSMsegmentproduct(s),
different regions of the putative M polypeptide
were expressed as glutathione S-transferase fusion
proteins, which were purified and inoculated into
mice to produce hyperimmune sera. By Western
blot analysis, a protein of approximately 30 kDa
and two glycoproteins, G1 and G2, with the same
molecular mass (approximately 65 kDa) were
identified in TOS virus-infected cells. The 30 kDa
protein, which reacted with antibodies raised to the
NH2-terminal, was found to be a non-structural
protein (designated NSm). By immunoprecipitation
analysis of TOS virus-infected cell lysates, both
treated or untreated with tunicamycin, the relative
positions of glycoproteins G1 and G2 were de-
termined. The gene order, with respect to the
genomic M RNA, was found to be 3? NSm-G1-G2 5?.
Toscana (TOS) virus is an enveloped negative-stranded
RNA virus with a segmented genome, and belongs to the
family Bunyaviridae, genus Phlebovirus. It was first isolated in
Italy from Phlebotomus perniciosus and P. perfiliewi sandflies
(Verani et al., 1984) and from cerebrospinal fluid in patients
with central nervous system diseases (Nicoletti et al., 1991).
Like other Bunyaviridae, it possesses three genomic segments
of ssRNA.The SRNA segment (1869 nucleotides)codes,in an
ambisense strategy, for the nucleocapsid protein (N; 27 kDa)
andanon-structural protein(NSs; 37 kDa)(Giorgiet al.,1991).
The L genomic segment (6404 nucleotides) can code for a
protein of 2095 amino acids, equivalent to 239 kDa (Accardi et
Author for correspondence: Colomba Giorgi.
Fax ?39 6 4990 2082. e-mail Giorgi?virus1.ISS.INFN.IT
The nucleotide sequence of the Toscana virus M genomic segment
has been submitted to the EMBL database and has been assigned
accession no. X89628.
al., 1993). A protein with a molecular mass greater than
200 kDa was identified in virus-infected cells by antibodies
directed against different regions of the L open reading frame
(ORF) (P. Di Bonito and others, unpublished results). In this
paper we report the coding organization of the M segment,
whichcompletesthecharacterizationoftheTOSviralgenome.
The genetic organization of the M RNA segment has been
reported for other phleboviruses – Rift Valley fever (RVF),
Punta Toro (PT) and Uukuniemi (UUK) viruses (Collett et al.,
1985; Ihara et al., 1985; Ro ? nnholm & Pettersson, 1987) – and
formany virusesbelonging toothergeneraof the Bunyaviridae
family (for review see Elliott et al., 1991; Schmaljohn, 1996).
Genetic and molecular analyses have demonstrated that the M
RNA segments of phleboviruses encode the envelope glyco-
proteins G1 and G2, and eventually one (PT) or two (RVF)
non-structural proteins. It has been predicted from sequencing
data that the M mRNA of phleboviruses has a single large
ORF,butthecorrespondingprecursorhasneverbeenidentified
in virus-infected cells, suggesting that it is processed co-
translationally. The maturation process of the M precursor
protein, and the enzymes involved, are not known. An
additional mechanism has been described for the biogenesis of
the two non-structural proteins of the RVF virus, identified
both in RVF virus-infected cells and in a cell-free system
(Kakach et al., 1989; Suzich & Collett, 1988). These proteins
seem to originate by differential translation starts at the first or
second AUG codon of the ORF (Suzich et al., 1990).
The TOS viral M segment was, in part, reconstructed from
specific clones identified in a cDNA library of TOS genomic
RNA (Giorgi et al., 1991). These clones covered the 5’-half of
the genomic segment. The 3?-half was obtained by RT–PCR
of viral nucleocapsid RNA, using oligonucleotides S12 (5?
CGAGAAGCTTACACAGAGA
sense, bases 1–9) and M4 (5? TTAAGCAGTCCGTGCA-
CACACAG 3?, viral sense, bases 2176–2156). Because the
sequences at the extremities of the phlebovirus genomic
segmentsareconserved,theS12oligonucleotidecontainedthe
eight bases complementary to the conserved bases of the TOS
S and L genomic 3? ends. The TOS M segment is 4215
nucleotides in length. The deduced amino acid sequence
identified only one long ORF, in the viral-complementary
sense sequence, which starts with an AUG codon at position
18–20 and terminates with a UAA termination codon at
3?,viral-complementary
0001-4138 ? 1997 SGM
HH

Page 2

P. Di Bonito and othersP. Di Bonito and others
208
144
87
44
32
123456
–
+–
++
–
TUN
G10
G20
208
144
87
44
32
17
123456
G1,G2
NSm
Anti-P1
Anti-P2
Anti-P3
(a)(b)
Fig. 1. (a) SDS–PAGE analysis of TOS virus-infected (lanes 3, 4, 5, 6) and uninfected (lanes 1, 2) cell lysates metabolically
labelled with [35S]Cys and either treated (lanes 2, 4, 6) or not treated (lanes 1, 3, 5) with tunicamycin (TUN). The lysates were
denatured at 37 ?C (lanes 3, 4) or 95 ?C (lanes 5, 6) before loading onto the gel. Positions of viral unglycosylated proteins
(G10and G20) and molecular mass markers are indicated on the right and the left, respectively. (b) Western blot analysis of
lysates from TOS virus-infected (lanes 2, 4, 6) and uninfected (lanes 1, 3, 5) cells. The samples were denatured at 37 ?C
before loading onto the gel. The membranes were tested with antibodies against the P1(Anti-P1), P2(Anti-P2) and P3(Anti-P3)
regions of the M ORF. The positions of the proteins identified (G1, G2 and NSm) and the molecular mass markers (kDa) are
indicated on the right and the left, respectively.
position4035–4037.Theencodedgeneproductis1339amino
acids in length, with a predicted molecular mass of 149 kDa.
The putative polypeptide is rich in cysteine residues (4%) and
has nine potential N-linked glycosylation sites.
To identify the product of the M segment, Vero cells were
mock-infected or infected with TOS virus at an m.o.i. of 1. At
8 h p.i. cells were incubated in cysteine (Cys)-free medium
containing tunicamycin, a known inhibitor of N-linked glyco-
sylation (Tkacz & Lampen, 1975) to 2 µg?ml. Labelling with
100 µCi?mlof[??S]Cys for1 h wascarriedoutat 10 hp.i.Cells
werethenlysedinSDSloadingbuffer(50 mMTrispH 6?8,3%
SDS, 5% β-mercaptoethanol, 10% glycerol) and the lysates
were analysed by SDS–PAGE.
As it has previously been reported that the bunyavirus G2
protein is extremely heat-sensitive (Madoff & Lenard, 1982;
Elliott, 1985; Gerbaud et al., 1992), the viral glycoproteins
were analysed by denaturing the cell lysate samples in loading
buffer at both 37 ?C and at 95 ?C. Under both conditions, a
viral protein with a molecular mass of about 65 kDa was
present in cell lysates from TOS virus-infected cells (Fig. 1a,
lanes 3 and 5, respectively). In lysates from virus-infected cells
treated with tunicamycin, two unglycosylated proteins (G1?
and G2?) were detected, rather than the 65 kDa protein, when
the samples were denatured at 37 ?C (Fig. 1a, lane 4). In the
95 ?C denatured samples (Fig. 1a, lane 6), the band cor-
responding to the slower migrating unglycosylated protein
(G1?) disappeared. This result clearly showed that the TOS
virus contains two glycoproteins (G1 and G2) which migrate
with the same electrophoretic mobility, and that G1 gly-
coproteinisheat-sensitive.Forthisreason,exceptwhenstated,
protein analysis was performed avoiding denaturation at
95 ?C.
To confirm this result and to localize the G1 and G2
proteins to the M ORF, three different regions of the putative
M-encoded protein were expressed in a prokaryotic system
and polyclonal antibodies were produced against them. DNA
fragments P?, P?and P?, corresponding to nucleotides
742–911, 1539–1893 and 3201–3526 of the viral comple-
mentary-sense nucleotide sequence of the TOS M segment,
were inserted into the multiple cloning site of the appropriate
pGEXvector(Pharmacia)inframewiththeCOOHterminusof
the glutathione S-transferase (GST) gene, under the control of
the IPGT-inducible tac promoter (Smith & Johnson, 1988). The
fusion proteins were recovered from cultures of bacterial cells
transformed with the recombinant plasmids and induced with
1 mM IPTG for 2 h. Clarified cell lysates were mixed with
glutathione–Sepharose beads (Pharmacia) and fusion proteins
were then eluted from the beads by competition with free
reduced-glutathione (Sigma). Swiss mice were then immunized
by three intraperitoneal injections (containing 10–20 µg of
protein per mouse each) at 2 week intervals. The animal sera
were tested for the production of specific antibodies by an
ELISA, using the fusion proteins as antigens. Three days after
the last boost, the ‘positive’ mice were injected with the
variant ascitic tumour cell line Sarcoma 180?TG to induce the
production of ascites in the animals (Beaty et al., 1989). The
ascitic fluids, collected 1 week later, were used as the source of
polyclonal antibodies.
Fig.1(b)showstheresultofaWesternblotanalysisofTOS
virus-infected cell lysates performed using the three different
HI

Page 3

Toscana virus M segmentToscana virus M segment
12341234
NSm
44
N
(a)
(b)
32
Fig. 2. Western blot using the anti-P1
antibodies. Proteins from purified virus
(lane 1), viral nucleocapsids (lane 2),
TOS virus-infected (lane 3) or uninfected
cells (lane 4) were separated on SDS–
polyacrylamide gel and then blotted onto
nitrocellulose membrane. The filters were
tested with mouse hyperimmune ascitic
fluid containing antibodies against the P1
fusion protein (a) and the N protein (b).
The specific proteins identified and the
molecular mass markers (kDa) are
indicated.
hyperimmune ascitic fluids. Cell lysates from uninfected and
virus-infected cells were run on an SDS–polyacrylamide gel.
Proteins were then transferred to a nitrocellulose membrane,
strips of which were incubated with the diluted hyperimmune
asciticfluid.Theantigen–antibodycomplexwasrevealedusing
peroxidase-conjugatedanti-mouseantibodies(Sigma)followed
by colorimetric analysis with 3,3?-diaminobenzidine tetra-
hydrochloride (DAB). The antibodies directed against the P?
andP?fusionproteinsreactedwith twoproteins with the same
electrophoretic mobility, confirming that the TOS virus
contains two glycoproteins of the same molecular mass. The
anti-P?antibodies recognized a protein with a molecular mass
ofabout30 kDa,whichusuallyappearedasadoubleband.The
anti-P?antiserum also reacted with different proteins with
molecular masses ranging approximately from 90 kDa to
130 kDa. It is unlikely that one of the bands represents the M
polyproteinprecursorbecauseitsmolecularmass,estimatedby
amino acid sequence (unglycosylated form), is 149 kDa. The
identity of these proteins is unclear. The P?region, positioned
at the putative COOH terminus of the NSm protein, probably
includes the peptide signal sequence of the next glycoprotein;
therefore, the anti-P?antiserum could react with potential
uncleaved precursors. However, the presence of these un-
cleaved precursors in TOS virus-infected cells was not
confirmed by either Western blot or immunoprecipitation
experiments with the anti-P?antiserum. On the other hand,
these bands could represent multimers of the 30 kDa protein,
or aggregates of this protein with cellular proteins, because
when the β-mercaptoethanol in the sample buffer was in-
creased, the intensity of these bands reduced (data not shown).
This protein has not been observed before in TOS virus-
infected cells, probably because in SDS–PAGE it runs with the
samemobilityastheabundantnucleocapsidproteinN.Forthis
reason we investigated its presence in viral nucleocapsids and
in mature virions. Fig. 2 shows a Western blot analysis of
proteins from cellular nucleocapsids (lane 1), purified virus
particles (lane 2), TOS virus-infected (lane 3) and uninfected
(lane 4) cells performed using the anti-P?antibodies (a) and
antibodies against the nucleocapsid N protein (b). The 30 kDa
protein was clearly revealed in TOS virus-infected cell lysates,
but not in viral nucleocapsids or in virus particles. This
indicates that the 30 kDa protein is a viral non-structural
protein and can be referred to as the non-structural protein of
the TOS M segment, NSm.
TodeterminetherelativepositionontheMsegmentofthe
unglycosylated forms of G1 and G2, immunoprecipitation
experiments were performed on the lysates from both
tunicamycin-treated and untreated cells infected with TOS
virus. The cells were labelled with [??S]methionine (Met) and
[??S]Cys as described above and lysed in RIPA buffer (50 mM
Tris–HCl pH 7?4, 150 mM NaCl, 1% Triton X-100,z 1 mM
EDTA, 0?1% SDS, 0?1% sodium deoxycholate and 0?6 trypsin
inhibiting units of aprotinin). Aliquots of cell lysates were
incubated with specific hyperimmune ascitic fluid and then
with protein A–Sepharose. The immunoprecipitated proteins
were eluted and analysed by SDS–PAGE (Fig. 3). The anti-P?
antiserum immunoprecipitated the NSm protein and two other
proteins with molecular masses of about 70 kDa and 130 kDa
(lane 1). In this experiment, as in Western blot analysis, the
identity of the proteins co-immunoprecipitated with NSm
remains unclear.
The anti-P?antiserum specifically immunoprecipitated a
single protein in lysates which had been either untreated (lane
5) or treated with tunicamycin (lane 6). We identified the
unglycosylated protein as G2?because its migration was
similartothatoftheheat-resistantviralunglycosylatedprotein
found in infected-cell lysates treated with tunicamycin (lane 4).
The anti-P?antibodies also immunoprecipitated a protein in
cell lysates (lane 7), but only in less stringent conditions
(0?025% SDS). In lysates from tunicamycin-treated cells, both
G1?and G2?were co-immunoprecipitated (lane 8), suggesting
that, under these experimental conditions the two proteins
couldinteracttooriginateheterodimers.Minorco-precipitated
bands, migrating as the viral N and NSs proteins, were also
visible.
The results reported in this study showed that, although
the TOS M segment possesses a single large ORF, it codes for
three proteins: a non-structural protein (NSm) and two major
glycosylatedproteins (G1 andG2) that areabout the samesize
(65 kDa). To identify the two major proteins, we took
advantage of the heat sensitivity of G1 and the different
electrophoretic mobility of their unglycosylated forms. By
using antibodies raised against specific portions of the putative
M polyprotein, the order of the proteins from the NH?
HJ

Page 4

P. Di Bonito and othersP. Di Bonito and others
––
–
+
–
+
–
+
144
87
44
32
G2
G20
NSs
N
G1
G20
G10
200
97·4
69
46
30
NSm
TUN
Anti-P1
Anti-P3
Anti-P2
78123456
Fig. 3. Immunoprecipitation analysis of lysates from TOS virus-infected cells metabolically labelled with [35S]Met and [35S]Cys.
The cells were treated with tunicamycin (TUN) or left untreated as indicated with ? or ?, respectively, at the top of the lanes.
The products immunoprecipitated by the anti-P1antibody from infected and uninfected cells are shown in lanes 1 and 2,
respectively. The ‘?’ symbols indicate the 65 kDa and 100 kDa proteins co-immunoprecipitated with the NSm protein. To
identify the immunoprecipitated product of the anti-P3antibody (lanes 5, 6), the total cell lysates, treated and untreated with
tunicamycin and denatured at 95 ?C, are shown in lanes 3 and 4. The position of the viral proteins NSs and N are shown for
these samples. G1 and G2 indicate the glycosylated proteins, whereas G10and G20indicate their unglycosylated forms.
Molecular mass markers (kDa) are also indicated.
terminus to the COOH terminus of the precursor protein was
shown to be NSm-G1-G2.
Lappin et al. (1994) proposed a new nomenclature for the
Bunyaviridae glycoproteins, following which the TOS G1
protein, located at the NH?terminus of the M precursor,
should be called G, and the G2 protein, at the COOH
terminus, G.
Wealsoidentifieda30 kDaproteinencodedatthegenomic
3?end.BothinWesternblotandRIPAexperimentstheprotein
usually migrated as a doublet. Since the two proteins were
recognized by antibodies raised against the presumptive
COOH region of NSm, the double band could have originated
by a differential translation, starting at different AUG codons,
or by different degrees of glycosylation of the same protein.
To distinguish between the two possibilities we examined the
NSm protein in virus-infected cells treated with tunicamycin
but, unfortunately, the anti-P?antibodies failed to recognize
the unglycosylated form of NSm protein (data not shown).
We showed that this protein is a non-structural protein
because it was not found in mature virions. Among the
phleboviruses, Uukuniemi virus lacks this protein and from
sequence data it is evident that the NH?-terminal region of the
polypeptides is the most variable region among the other
members of the genus. This fact is reminiscent of the non-
structural proteins of the S segments of these viruses, NSs,
which do not show any sequence similarities among the
phleboviruses. The exact location and function of these
proteins are still unknown, but they could play a distinctive
role in the biological properties of the different phleboviruses.
We would like to thank Mrs Giulia Pacetto and Sabrina Tocchio for
editorial assistance, and Michelle Concra for linguistic revision of the
manuscript.
References
Accardi, L., Gro ? , M. C., Di Bonito, P. & Giorgi, C. (1993). Toscana virus
L segment: molecular cloning, coding strategy and amino acid sequence
in comparison with other negative strand RNA viruses. Virus Research27,
119–131.
Beaty, B. J., Calisher, C. H. & Shope, R. E. (1989). Arboviruses. In
Diagnostic Procedures for Viral, Rickettsial and Clamydial Infections, 6th edn,
pp. 797–855. Edited by N. J. Schmidt & R. W. Emmons. Washington,
DC: American Public Health Association.
Collett, M. S., Purchio, A. F., Keegan, K., Frazier, S., Hays, W.,
Anderson, D. K., Parker, M. D., Schmaljohn, C., Schmidt, J. &
Dalrymple, J. M. (1985). Complete nucleotide sequence of the M RNA
segment of Rift Valley fever virus. Virology 144, 228–245.
Elliott, R. M. (1985). Identification of nonstructural proteins encoded by
viruses of the Bunyamwera serogroup (family Bunyaviridae). Virology
143, 119–126.
Elliott, R. M., Schmaljohn, C. S. & Collet, M. S. (1991). Bunyaviridae
genome structure and gene expression.Current Topics in Microbiology and
Immunolology 169, 91–141.
Gerbaud, S., Pardigon, N., Vialat, P. & Bouloy, M., (1992). Organ-
ization of Germiston bunyavirus M open reading frame and physico-
IA

Page 5

Toscana virus M segmentToscana virus M segment
chemical properties of the envelope glycoproteins. Journal of General
Virology 73, 2245–2254.
Giorgi, C., Accardi, L., Nicoletti, L., Gro ? , M. C., Takehara, K., Hilditch,
C., Morikawa, S. & Bishop, D. H. L. (1991). Sequences and coding
strategies of the S RNAs of Toscana and Rift Valley fever viruses
compared to those of Punta Toro, Sicilian sandfly fever and Uukuniemi
viruses. Virology 180, 733–753.
Ihara, T., Smith, J., Dalrymple, J. M. & Bishop, D. H. L. (1985).
Complete sequences of the glycoprotein and M RNA of Punta Toro
phlebovirus compared to those of Rift Valley fever virus. Virology 144,
246–259.
Kakach, L. T., Suzich, J. A. & Collett, M. S. (1989). Rift Valley fever
virus M segment: phlebovirus expression strategy and protein glycosyl-
ation. Virology 170, 505–510.
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. Journal of General
Virology 75, 3441–3451.
Madoff, D. M. & Lenard, J. (1982). A membrane glycoprotein that
accumulates intracellularly: cellular processing of a large glycoprotein of
La Crosse virus. Cell 28, 821–829.
Nicoletti, L., Verani, P., Caciolli, S., Ciufolini, M. G., Renzi, A.,
Bartolozzi, D., Paci, P., Leoncini, F., Padovani, P., Traini, E.,
Baldereschi, M. & Balducci, M. (1991). Central nervous system
involvement during infections by phlebovirus Toscana of residents in
natural foci in Central Italy (1977–1988). American Journal of Tropical
Medicine Hygiene 45, 429–434.
Ro?nnholm, R. & Pettersson, R. F. (1987). Complete nucleotide
sequence of the M RNA segment of Uukuniemi virus encoding the
membrane glycoproteins G1 and G2. Virology 160, 191–202.
Schmaljohn, C. S. (1996). Bunyaviridae: thevirusesand theirreplication.
In Fields Virology, pp. 1447–1471. Edited by B. N. Fields, D. M. Knipe &
P. M. Howley. Philadelphia: Lippincott-Raven.
Smith, D. B. & Johnson, K. S. (1988). Single-step purification of
polypeptides expressed in Escherichia coli as fusion with glutathione S-
transferase. Gene 67, 31–40.
Suzich, J. A. & Collett, M. S. (1988). Rift Valley virus M segment: cell
free transcription and translation of virus-complementary RNA. Virology
164, 478–486.
Suzich, J. A., Kakach, L. T. & Collett, M. S. (1990). Expression strategy
of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus
M segment. Journal of Virology 64, 1549–1555.
Tkacz, J. S. & Lampen, J. O. (1975). Tunicamycin inhibition of
polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf
livermicrosomes.BiochemicalandBiophysicalResearchCommunications65,
248–257.
Verani, P., Nicoletti, L. & Ciufolini, M. G. (1984). Antigenic and
biological characterization of Toscana virus, a new phlebotomus fever
group virus isolated in Italy. Acta Virologica 28, 39–47.
Received 19 May 1996; Accepted 18 September 1996
IB