Download Structure, expression and phylogenetic analysis of the glycoprotein

Virus Research 54 (1998) 197 – 205
Short communication
Structure, expression and phylogenetic analysis of the
glycoprotein gene of Cocal virus
Resham S. Bhella 1,a, Stuart T. Nichol b, Essam Wanas a, Hara P. Ghosh a,*
b
a
Department of Biochemistry, McMaster Uni6ersity, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
Special Pathogens Branch, Di6ision of Viral and Rickettsial Diseases, Centers for Disease Control and Pre6ention, Atlanta,
GA 30333, USA
Received 8 October 1997; received in revised form 4 December 1997; accepted 10 December 1997
Abstract
A cDNA copy of the mRNA of the glycoprotein G of Cocal virus, a rhabdovirus, has been cloned, sequenced and
expressed in mammalian cells. The deduced amino acid sequence shows a typical transmembrane glycoprotein, 512
amino acids in length, containing two potential N-linked glycosylation sites. The amino acid sequence showed a high
degree of identity with that of the prototype vesicular stomatitis virus serotype Indiana [VSV (IND)] G protein. In
addition, phylogenetic analysis of amino acid sequence differences among the G proteins of vesiculoviruses indicated
that Cocal virus represents a distinct lineage within the VSV (IND) serotype. Expression of the cloned Cocal G gene
in mammalian cells produced a glycoprotein of mol.wt 71000 which was not palmitylated but induced cell fusion at
acid pH. © 1998 Elsevier Science B.V. All rights reserved.
Keywords: Glycoprotein G; Cocal virus; Vesicular stomatitis; Rhabdovirus
Glycoproteins of enveloped animal viruses have
been used extensively to study biogenesis, transport and targeting of membrane glycoproteins
(Ghosh, 1980; Garoff, 1985; Einfeld and Hunter,
* Corresponding author. Tel.: +1 905 5259140, ext. 22451;
fax: +1 905 5229033; e-mail: [email protected]
1
Present address: NRC Plant Biotechnology Research Institute, Saskatoon, Saskatchewan, Canada.
1991; Pettersson, 1991). The single glycoprotein G
of vesicular stomatitis virus of the Indiana
serotype [VSV (IND)], which is the prototype of
the negative-stranded rhabdovirus family, has
been used as a model in many of these studies.
Since the G protein plays a central role in virus
entry and assembly of infectious particles as well
as antigenicity and host range determination,
identification of the various functional domains of
0168-1702/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.
PII S0168-1702(98)00006-9
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this protein is essential to understand the structure-function relationship of this protein (Wagner,
1990). A knowledge of the sequences of G
proteins of other serological strains of VSV as
well as temperature sensitive mutants, is also required to examine evolutionary relationship and
antigenic diversity of these viruses (Bilsel and
Nichol, 1990). The G protein genes of three vesiculoviruses other than VSV (IND), namely VSV
New Jersey serotype [VSV (NJ)], Chandipura
(CHP) and Piry have been sequenced (Rose and
Gallione, 1981; Gallione and Rose, 1983; Masters
et al., 1989, 1990; Brun et al., 1995). Cocal virus
was originally isolated from mites in Trinidad
(Jonkers et al., 1984), but infections have since
been detected in Trinidad, Brazil and Argentina
including isolation of the virus from cattle, horses,
and insects (Karabatsos, 1985). The virus is serologically distinct but related to the VSV (IND)
prototype strain (Jonkers et al., 1984; Travassos
da Rosa et al., 1984). We report here, the complete sequence of the G protein gene of Cocal
virus. The cloned gene expresses a biologically
active G protein.
Cocal virus was grown in monolayers of mouse
L or baby hamster kidney (BHK-21) or HeLa
cells. A full length cDNA clone of the G mRNA
of Cocal virus was generated from poly A+
mRNA using the protocol of Gubler and Hoffman (1983). The cDNA was cloned in the EcoRI
site of pGEM7Zf(+) and finally into the COS
cell expression vector pXM (Yang et al., 1986).
DNA sequence was determined by using dideoxy
chain termination method using a modified T7
DNA polymerase (Sequenase) (Sanger et al.,
1977) and nested deletion of nucleotides
(Henikoff, 1984). The sequence was reconfirmed
by sequencing the DNA of the gene cloned in
pXM [pXMG(COC)] using synthetic oligonucleotide primers. Genomic RNA extracted from Cocal virions was also sequenced to determine
sequences of nucleotides 1 – 300, 971 – 1140 and
1460 –1652 by using oligonucleotide primers and
avian myeloblast virus reverse transcriptase (Bilsel
and Nichol, 1990).
The complete nucleotide sequence of the G
gene was determined (Fig. 1) and deposited in the
Gene Bank, EMBL. The Cocal G mRNA spans
1602 nucleotide excluding the poly(A) tail. It contains a single continuous open reading frame
starting at the first ATG at 29 nucleotides from
the 5%-end. The encoded protein contains 512
amino acids with a calculated mol.wt of 58036,
which is in agreement with its observed mobility
in SDS-polyacrylamide gels (Kotwal et al., 1983).
In agreement with other vesiculovirus mRNAs,
the G mRNA has the following terminal
sequences:
5% AACAG(N)23AUG-A coding region - UGAUGA(N)28UAUGA7 –poly(A) 3%
The sequence 3% UUGUC 5% represents the
vesiculovirus conserved transcription initiation
(capping) sequence (Banerjee and Barik, 1992),
while 3% AUACU7 5% represents the transcription
termination and polyadenylation signal in the viral genome (Banerjee and Barik, 1992). As in the
case of VSV (IND) genome the G-L intragenic
sequence of Cocal virus 5% UAUGA7CUAACAG
3%, did not contain any insertion of nucleotides
(Rose, 1980). The 5% AACAG 3% at the 3% end of G
mRNA denotes the transcription initiation signal
for the L gene. The translation initiator methione
codon AUG like the other three vesiculovirus G
proteins, also contains a strong eukaryotic ribosomal initiation context (Kozak, 1989).
The deduced amino acid sequence of Cocal G
protein (Fig. 2) was compared with the amino
acid sequence of the N-terminal 24 residues of the
mature glycoprotein isolated from Cocal virions
or from the infected cells as well as with the
amino acid sequence of the primary translation
product of the G mRNA (Kotwal et al., 1983).
The results showed identity between the deduced
sequence with the sequence determined in both
the signal sequence and in the sequence of the
processed mature G protein. The predicted signal
sequence of 17 residues was identical to the signal
peptide determined from direct sequencing except
for two residues: a Lys and Leu were determined
at positions − 16 and − 11, respectively, instead
of Asn and Thr residues predicted in the corresponding position. The N-terminal 24 residues
were identical except for the fact that at position
22 the virion protein contained Tyr while the
deduced sequence predicted His. These differences
could be accounted for by one or two base
R.S. Bhella et al. / Virus Research 54 (1998) 197–205
199
Fig. 1. Complete nucleotide sequence of glycoprotein G gene of Cocal virus. The conserved vesiculovirus transcription signals and
transcription termination and polyadenylation signals (Banerjee and Barik, 1992) are underlined. The translation initiation and
termination codons are indicated by arrowheads.
changes occurring either during the cloning of the
cDNA or at the virus genome as a result of
random mutations produced during serial passages (Wagner, 1990).
In order to ascertain the biological activity of
the cloned G gene, COS cells were transfected
with the plasmid pXMG(COC), an eukaryotic
expression vector containing the complete coding
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R.S. Bhella et al. / Virus Research 54 (1998) 197–205
Fig. 2. Alignment of the deduced amino acid sequences of the G proteins of Cocal, VSV Indiana serotype (IND) (Rose and
Gallione, 1981), VSV New Jersey serotype (NJ) (Gallione and Rose, 1983), Chandipura (CHP) (Masters et al., 1989, 1990; Brun et
al., 1995) and Piry (Brun et al., 1995) viruses. Dashed lines represent gaps introduced to maximize matching of amino acid residues.
Residues not shown and shaded are identical to those of Cocal G protein. Shaded residues indicate conservative changes. The arrow
indicates the signal peptide cleavage site. The asterisk above N indicates consensus site for N-linked glycosylation. The hydrophobic
membrane anchoring domain is underlined. The conserved Cys, Pro and Trp residues are indicated with a circle.
R.S. Bhella et al. / Virus Research 54 (1998) 197–205
201
Fig. 3. Expression, glycosylation and fatty acid acylation of Cocal G protein from cloned gene. (A) COS cells were transfected with
pXMG(IND), pXMG(COC) or no DNA and labelled with [35S]methionine. The cell lysates were immunoprecipitated with anti-VSV
Indiana G for pXMG(IND) transfected cells and anti-Cocal G antibody for cells transfected with pXMG(COC) or no DNA and
analyzed by SDS-polyacrylamide gel electrophoresis (Li et al., 1993). (B) N-linked glycosylation of expressed Cocal G proteins was
determined by the acquisition of endo H resistance (Li et al., 1993). Proteins expressed in cells transfected with pXMG(COC) were
labeled with [35S]methionine for 15 min, chased with excess of non-radioactive methionine for a period of 0 min or 60 min, and
immunoprecipitated with anti-Cocal G antibody. One half of the sample was treated with endo H ( +), and the other half was not
(−). Samples were analyzed by SDS-polyacrylamide gel electrophoresis. (C) COS cells transfected with pXMG(COC) or
pXMG(IND) were labeled with [3H]palmitic acid (Kotwal and Ghosh, 1984; Masters et al., 1989), immunoprecipitated with
anti-Cocal G or anti-VSV Indiana G antibody, respectively, and analyzed on SDS-polyacrylamide gel.
region of Cocal G gene. Transfected cells were
labelled with [35S]methionine and labelled proteins
were immunoprecipitated with anti-Cocal G antiserum. As shown in Fig. 3A, the antibody recognized a protein of about 71000 Da, the expected
size of Cocal G protein (Kotwal et al., 1983). This
protein was not present in an immunoprecipitate
of COS cells transfected with the parent vector
pXM. The enzyme endoglycosidase H (endo H)
hydrolyzes the oligomannose moiety of N-linked
glycoproteins containing unprocessed glycosyl
residues, while the mature glycoproteins containing processed and complex oligosaccharide
residues are resistant to endo H digestion (Kornfeld and Kornfeld, 1985). Digestion of the expressed protein with endo H shows that the
immature G protein is sensitive to endo H but the
mature G protein produced after 1-h chase is
resistant to endo H digestion (Fig. 3B) indicating
that Cocal G protein expressed from cloned G
contained N-linked oligosaccharide. This is in
agreement with the predicted Cocal G protein
amino acid sequence, which shows the presence of
two N-linked glycosylation sites at residues 180
and 337. Similar to the results obtained with
Cocal virus infected cell (Kotwal and Ghosh,
1984), no palmitylation of the expressed G protein
was also observed in the transfected COS cells,
which is consistent with the absence of any Cys
residues in the membrane anchor or cytoplasmic
domains of Cocal G protein (Fig. 3C). The
G(IND) protein was, as reported earlier, labeled
with palmitic acid. As expected, the expressed G
protein was also transported to the cell surface as
determined by indirect immunofluorescence (Fig.
4A).
Glycoprotein G of VSV expressed from cloned
gene can induce membrane fusion at acidic pH in
the absence of other viral gene products
(Florkiewitz and Rose, 1984; Reidel et al., 1984).
When COS cells expressing Cocal G protein were
briefly exposed to a buffer of pH 5.6, polykaryons
containing more than 20 nuclei were observed
(Fig. 4B). This demonstrated that, like the G
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Fig. 4. Cell surface localization and polykaryon formation by Cocal G protein. (A) COS cells transfected with pXMG(COC) or no
DNA were fixed with paraformaldehyde and reacted with rabbit anti-Cocal G antiserum and fluorescein isothiocynate-conjugated
anti-rabbit immunoglobulin G (Zhang and Ghosh, 1994). (B) COS cells transfected with pXMG(COC) were exposed to fusion
medium at pH 7.4 or 5.6 for 60 s, incubated in regular medium for 2 h, re-exposed to fusion medium at pH 7.4 or 5.6 for 60 s,
incubated in regular medium for 1.5 h, fixed, stained and photographed (Zhang and Ghosh, 1994).
protein of VSV (IND) (Florkiewitz and Rose,
1984; Reidel et al., 1984) or Chandipura (Masters
et al., 1989), the G protein of Cocal virus showed
low-pH dependent fusogenic property (Kotwal et
al., 1983).
In order to locate functionally homologous and
unique domains in the G protein, we have aligned
and compared the deduced sequences of the G
proteins of VSV (IND) (Rose and Gallione,
1981), CHP (Masters et al., 1989, 1990; Brun et
al., 1995), VSV (NJ) (Gallione and Rose, 1983),
and Piry (Brun et al., 1995) with that of Cocal
virus (Fig. 2). The sequence alignment shows that
G protein of Cocal virus has about 48, 35 and
38% identity to that of G proteins of VSV (NJ),
CHP and Piry, respectively. However, the identity
of sequence of Cocal G with that of VSV (IND)
G protein was 72%, and when conservative substitutions are included the similarity increased to
77%. Also, the G protein sequence alignment
contains a large number of blocks of amino acids
identical between the five viruses (Fig. 2).
The following important structural features of
Cocal G protein may thus be tentatively
identified.
(i) The signal sequence of Cocal G was previously determined by us (Kotwal et al., 1983) to be
one amino acid longer than the signal sequence of
G protein of VSV (IND) or VSV (NJ). The
predicted signal sequence and the site of cleavage
were identical to those determined by direct
amino acid sequence determination (Kotwal et al.,
1983), and N-terminal Lys residue was present in
mature G proteins of VSV (IND), VSV (NJ) and
Cocal (Kotwal et al., 1983).
(ii) The two N-linked glycosylations occur at
identical positions on all five G proteins.
(iii) The membrane anchoring sequence of hydrophobic amino acids is located at positions
465–483 of Cocal G protein. Although the
transmembrane sequences of glycoproteins of related virus usually do not show homology, the
R.S. Bhella et al. / Virus Research 54 (1998) 197–205
sequence of the transmembrane domain of Cocal G protein shows a 53% identity with that
of VSV (IND) G protein.
(iv) The cytoplasmic domain of Cocal and
the other four vesiculovirus G proteins showed
very little conservation. However, the cytoplasmic sequence was shown to be essential in virus
assembly presumably by recognition of the nucleoprotein complex (Whitt et al., 1989).
(v) Three amino acids, Trp, Pro and Cys, are
found to occur in non-variant positions in the
G proteins of all five vesiculoviruses. Conserved
Pro residues could define domains while the
conserved Cys residues are possibly involved in
disulfide bond formation essential for proper
folding. It may be noted that no Cys residues
are present after amino acid 301.
(vi) Several blocks of amino acids are also
conserved in all five sequences. The function of
the conserved 17-amino acid stretch spanning
residue 83–99 is not yet known. The conserved
region spanning residue 124 – 137 has recently
been identified as a fusogenic domain of G
protein of VSV (IND) (Li et al., 1993; Zhang
and Ghosh, 1994; Durrer et al., 1995; Frederickson and Whitt, 1995). This region may thus
represent the fusion peptide of vesiculovirus G
proteins. A conserved region encompassing
residues 395–424 near the membrane anchoring
domain of VSV (IND) G protein has also been
recently shown to be important for membrane
fusogenic activity of G protein and may play a
key role in control of low-pH induced conformational change of rhabdovirus glycoproteins
(Li et al., 1993; Gaudin et al., 1996; Shokralla
et al., 1998). The functional role of the other
conserved blocks containing 4 – 6 amino acids is
yet to be established. Mutagenesis in the conserved regions of G protein and the effect of
the mutations on biological activities of G
protein may provide some information on the
structure-function relationship of G proteins.
(vii) Finally, phylogenetic analysis of the
amino acid sequence differences among representative vesiculoviruses (Fig. 5) clearly indicates that Cocal virus is closely related to VSV
(IND) but represents a distinct lineage separate
from classical VSV (IND) strains. This corre-
203
Fig. 5. Phylogenetic relationship of vesiculoviruses based on
analysis of G protein deduced amino acid sequence differences. Phylogenetic analysis was performed by the maximum
parsimony method using PAUP 3.1 software (Swofford, 1991).
The analysis was done using the Branch and Bound (BANDB)
and MULPARS options, with PROTPARS weighting of the
data matrix (Felsenstein, 1993), and bootstrap confidence limits obtained from 1000 repetitions of the analysis. The more
distantly related vesiculovirus, Chandipura virus, G protein
sequence was used to outgroup root the tree output. The
horizontal distances represent the number of amino acid step
differences (indicated by scale bar) present between branch
nodes and taxa (i.e. viruses). Vertical distances are for graphic
representation only. Bootstrap confidence limits exceeding
50% are indicated next to each branch node.
lates with previous findings showing the serological cross-reactivity of Cocal virus with a
VSV (IND) serotype prototypic virus isolate
(Travassos da Rosa et al., 1984).
Acknowledgements
We thank Xiaynan Li for technical assistance, Pamuk Bilsel for preliminary sequences
and L. Kush and M.M. Strong for manuscript
preparation. This work was supported by Medical Research Council of Canada (H.P.G.).
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