key: cord-0752193-eaj63c2k
authors: Matsuoka, Yumiko; Ihara, Takeshi; Bishop, David H.L.; Compans, Richard W.
title: Intracellular accumulation of punta toro virus glycoproteins expressed from cloned cDNA
date: 1988-11-30
journal: Virology
DOI: 10.1016/0042-6822(88)90075-x
sha: 526922ed72fc135d77d546ebee9e38f414f12ba4
doc_id: 752193
cord_uid: eaj63c2k

Abstract The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NSM. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NSM sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.

The family Bunyaviridae comprises a large and heterogeneous group of arthropod-borne viruses with certain structural features in common (Bishop and Shope, 1979; Bishop et a/., 1980) . The viruses are enveloped, and contain two surface glycoproteins, Gl and G2, an internal nucleocapsid-associated protein N, and a large internal protein (L) believed to be a transcriptase component. The nucleocapsid

consists of three singlestranded RNA genome segments, L, M, and S. On the basis of genetic recombination and sequence analysis, it has been concluded that the S RNA encodes the nucleocapsid protein N and a nonstructural protein NS,; the M RNA encodes two glycoproteins Gl and G2 and in some genera a nonstructural protein, NS,; and the L RNA probably contains the information for the viral transcriptase (Bishop et al., 1982; Bouloy et a/., 1984; Cabradilla et al., 1983; Collett et a/., 1985; Eshita and Bishop, 1984; Fuller et al., 1983; lhara et a/., 1985; Lees et al., 1986; Ronnholm and Pettersson, 1987; Schmaljohn et a/., 1987 , 1982, 1984) .

' To whom requests for reprints should be addressed.

It has also been demonstrated by using temperaturesensitive mutants that the intracellular accumulation of glycoproteins occurs in the absence of virus maturation (Gahmberg er a/., 1986). lntracytoplasmic virus budding has also been described for coronaviruses (Dubois-Dalcq eta/., 1982; Holmes et al., 1981; Tooze et a/., 1984; Tooze and Tooze, 1985) , flaviviruses (Lear-y and Blair, 1980) , toroviruses (Fagerland et a/., 1986; Weiss and Horzinek, 1986) , and rotaviruses (Altenburg et al., 1980; Petrie et al., 1984) . It has been reported that the El glycoprotein of the coronaviruses, when expressed from cloned DNA, accumulated intracellularly (Machamer and Rose, 1987; Rottier and Rose, 1987) . Furthermore, evidence has recently been reported which indicates that one of three hydrophobic membrane-spanning domains of this protein is responsible for retention in the Golgi complex (Machamer and Rose, 1987) .

Punta Toro virus (PTV) is one of some 36 arthropodborne viruses assigned to the Phlebotomus fever serogroup (Phlebovirus genus, Bunyaviridae, Bishop et a/., 1980) and has structural and morphological features similar to other members of the family Bunyaviridae. The S RNA of PTV has an unusual ambisense coding strategy. One protein (N) is coded in a subgenomic, viral-complementary S mRNA species and a second protein (NSs) is coded by a viral sense mRNA (Ihara et a/., 1984) . The M RNA segment possesses a single open reading frame in the viral-complementary sequence that is presumed to code for a polyprotein precursor.

In addition to the Gi and G2 glycoprotein coding sequences, a sequence capable of coding a polypeptide of approximately 30 kDa was found preceding the Gi coding sequences, although the putative NSM protein product corresponding to this sequence has not been identified either in virions or in infected cells. The estimated sizes of N&, Gl , and G2 from the predicted amino acid sequence are 30,510, 60,897, and 55,005 Da, respectively (Ihara et a/., 1985) . We have constructed vaccinia virus recombinants expressing the Gl and G2 glycoproteins of PTV, in order to study the possible existence of signals for intracellular retention in bunyavirus glycoproteins.

We report here the expression of the two glycoproteins of PTV, and their localization in infected cells in the absence of other viral components.

CV-1, Vero, and HeLaT4+cells(Maddoneta/., 1986) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn bovine serum. Human TK-143 cells maintained in the above medium supplemented with 25 pg/ml of 5bromo-2-deoxyuridine (5-BUdR). Vaccinia virus stocks were prepared and titrated by plaque assay in CV-1 cells. PTV stocks were prepared and titrated in Vero cells.

of a vaccinia recombination plasmid containing the Gl and G2 glycoprotein genes All DNA manipulations were carried out as described by Maniatis et a/. (1982) . Four cDNA clones, 5-79, 1 O-99, 6-37, and 4-148, which were previously used for sequence analysis, had been cloned into the Pstl site of pBR322 (Ihara et a/., 1985) . By a series of restriction digestions and ligations with these clones, a full-length PTV M cDNA clone was constructed and inserted into pGEM-3 (Promega Biotech, Madison, WI), designated as pGEM-M. It was found, however, by analysis of protein synthesis in an in vitro transcription and translation system using pGEM-M and by subsequent sequence analysis that pGEM-M contained deletion mutations in the middle of the Gl glycoprotein coding region. It was also found that the sequences preceding the Gl coding sequences seem to have some inhibitory effects on growth of bacterial colonies. Because of these problems, most of the 5'sequence preceding the Gl coding region was removed by digestion with Ba13 1 exonuclease. In order to repair the mutations, the Ncol-Xbal restriction fragment which contains the mutated region was replaced by the corresponding fragment from clone 5-79, one of the original clones used for sequence analysis (Fig. 1) . After transformation and colony selection, two clones were obtained, one of which contained an M fragment beginning with nucleotide residue 702 that had an ATG at nucleotide residue 716, whereas the other contained a fragment beginning with residue 765 and lacked an initiation codon for synthesis of G 1 and G2 glycoproteins.

The clone containing an ATG codon in the region preceding the G! coding sequences was designated as pGEM-G(C) since it possesses a C residue at the -3 position upstream from the translation initiation site. The clone which did not have an ATG sequence was further modified to introduce a translation initiation codon. After digestion with EcoRI and treatment with Klenow DNA polymerase, C/al linkers were added at the ends of the DNA in order to provide an ATG sequence. This segment was then digested with C/al and treated with Klenow polymerase following digestion with either Pstl or SalI in order to remove plasmid sequences.

These segments were inserted into either pGEM-3 digested with Sal1 and treated with Klenow polymerase following Pstl digestion or pGEM-3 digested with Xbal and treated with Klenow polymerase following SalI digestion. As a result, pGEM-G(A) which contains an A residue at the -3 position from the initiation codon, and pGEM-G(G) with a G residue at the -3 position, were obtained (Fig. 1 ).

The PTV glycoprotein gene was excised by BarnHI from the three clones, pGEM-G(C), pGEM-G(A), and pGEM-G(G), treated with Klenow polymerase, and inserted into the Smal site of the pSC1 1 vaccinia recombination plasmid (Chakrabarti et a/., 1985) .

of a vaccinia recombination plasmid containing the influenza HA gene

The cDNA clone of the WSN-HA gene in pBR322 was kindly provided by Dr. Debi Nayak. The HA gene was excised from pBR322 and recloned into the Pstl site of pUC13 (Stephens et al., 1986) . The plasmid was liniarized by BamHl digestion, treated with Ba/31 exonuclease to remove dG and dC homopolymeric tails at the 5' end, treated with Klenow DNA polymerase, and then digested with HindIll. The HA gene fragment was separated from the pUCl3 vector, inserted into HindIll-Hincll-digested pGEM-3, excised byXbal-HindIll digestion, treated with Klenow DNA polymerase, and inserted into the Smal site of pSCl1. , 1983) . To select for recombinants, TK-143 cells were infected with 50-l 00 PFU of TK-vaccinia virus in the presence of BUdR (25 pg/ml). At 48 hr after infection, the monolayers were overlayed with 19/o low-melting-point agarose containing 300 pg/ml of 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-Gal). AT 4-6 hr, blue plaques were picked and further purified by two additional rounds of plaque purification.

Hyperimmune mouse ascitic fluids specific for PTV and monoclonal antibodies against Gl and G2 glycoproteins were kindly provided by Drs. J. F. Smith and D. Pifat (USAMRIID, For Petrick, MD). Rabbit antiserum against A/WSN/33 influenza virus was prepared as described elsewhere (Roth and Compans, 1980) . CV-1 cells or HeLa T4+ cells were infected with vaccinia virus recombinants or PTV at an m.o.i. of 5 to 10. At 16 hr p.i., cells were washed with phosphate-buffered saline (PBS) and incubated in methionine-free medium for 2 hr. At 18 hr p.i., cells were labeled with [35S]methionine (100 &i/ml) in methionine-free medium for 1 hr. For pulse-chase experiments, cells were pulsed for 10 min and then chased in Eagle's medium containing 10 mlVl methionine for an appropriate period. Cells were then washed three times in ice-cold PBS and lysed with 0.3 ml of lysis buffer (50 mM Tris-HCI (pH 7.5), 0.15 M NaCI, 1 o/o Triton X-l 00, 0.1 Oh SDS, 20 mM EDTA). Nuclei were removed by centrifugation at 13,000 g for 5 min at 4". The cell lysates were incubated with 5 /-cl of mouse ascites against PTV for 4 hr at 4" with constant mixing. For immunoprecipitation of cell-surface antigens, antibody was added before cells were lysed and incubated for 30 min on ice. After washing to remove unbound antibody, radiolabeled polypeptides were immunoprecipitated with protein A-Sepharose CL-4B (Pharmacia, Inc., Piscataway, NJ). The precipitates were washed three times with cold lysis buffer, resuspended in sample buffer, boiled for 5 min, and analyzed on 10% SDS-polyacrylamide gels (Laemmli, 1970) .

HeLa T4+ During the labeling period, PMSF and TM were maintained in medium of treated cells. Half of each sample was further treated by endo H at 37" for 16 hr (lanes b, d, f, h, and j). All samples were then analyzed on 10% SDS-PAGE.

Laboratories, Inc., San Mateo, CA) in some experiments. The cells were washed with PBS mounted, and observed with a Nikon Optiphot microscope equipped with a modified B2 cube.

H digestion lmmunoprecipitated samples were boilded for 5 min in SDS-PAGE sample buffer and the protein A-Sepharose was removed. The supernatants were diluted 15fold with 0.1 M sodium acetate, pH 6.0, and incubated for 16 hr at 37" in the presence of 8 mu/ml of endoglycosidase H (endo H) (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and 1 mM of PMSF. Samples were then precipitated with lOoh TCA for 1 hr at O", washed with cold ethanol/ether (1 :l), and resuspended for SDS-PAGE analysis.

To quantitate antigen expressed on cell surface, surface radioimmunoassays were performed by a method described by Stephens er al. (1986) . HeLa T4+ cells on glass coverslips were infected with vaccinia virus or the vaccinia virus recombinant at an m.o.i. of 10. At appropriate times after infection, cells were washed three times with PBS/l % bovine serum albumin (BSA), overlaid with 10 ~1 of antibody diluted 1: 10 in PBS/l % BSA, and incubated for 30 min at 4". Cells were then washed three times with PBS/l% BSA and incubated with 200,000 cpm of '251-protein A (Amersham, Arlington Heights, IL) prepared in PBS/is/o BSA for 30 min at room temperature. The label was removed, the cells were washed three times with PBS/l% BSA, and the bound radioactivity was determined. Nonspecific binding of the '251-protein A to cells infected with wild-type vaccinia virus was determined for each antisera and subtracted from each value.

To compare the levels of PlV glycoprotein synthesis, monolayers of CV-1 cells were infected with wildtype vaccinia virus, PTV, or the vaccinia recombinants described above which contain the PTV glycoprotein gene with the different sequences in the translation initiation region. At 18 hr p.i., cells were labeled for 1 hr with [35S]methionine, harvested, and immunoprecipitated. Two polypeptides, which have electrophoretic mobility similar to those of the Gl and G2 glycoproteins synthesized in PTV-infected cells, were precipitated from cells infected with vaccinia recombinants using antibody specific for PTV ( Fig. 2A) . The polypeptide corresponding to the G2 glycoprotein occasionally appeared as a doublet. The appearance of the lower band was not consistently observed, and it may be a differently glycosylated form of the G2 glycoprotein. A similar observation of the synthesis of a heterogeneous Hantaan (HTN) Gl glycoprotein was reported by Pensiero et a/. (1988) . The level of protein synthesis was much higher for the recombinants which contain G or A at the -3 translation initiation position than the one which contained C. Therefore, one of the recombinants which contains G at the -3 position was chosen as a prototype (designated VV-G) and used for further experiments.

The effect of tunicamycin (TM) on the synthesis of the proteins was also examined as shown in Fig. 2B . At 16 hr p.i., cells infected with PTV or recombinants were exposed to medium containing TM for 2 hr. Cells were then labeled with [35S]methionine for 1 hr, lysed, and immunoprecipitated.

The Gl glycoproteins synthesized from the VV-G recombinant, in eitherthe presence or absence of TM, showed slightly slower mobility on the gel then those synthesized in PTV-infected cells. The G2 glycoprotein appeared as multiple bands in the presence of TM, which may indicate a partial inhibition of glycosylation.

The amount of Gl and G2 glycoproteins detected in the presence of TM was significantly lower than that in the absence of TM. In contrast, under the same condition, the glycosylation of influenza HA glycoprotein was completely inhibited and similar amounts of both glycosylated and unglycosylated forms of HA were detected. When a higher concentration of TM (1.5 pg/ml) was used, neither the Gl nor G2 glycoprotein was detected (data not shown). Therefore, the unglycosylated forms of Gl and G2 glycoproteins appear to be relatively unstable and quickly degraded even in the presence of PMSF.

The intracellular localization of the expressed glycoproteins was examined in HeLa T4+ cells because this cell line shows minimal cytopathic effect with vaccinia virus infection when compared to most other cell lines, and is therefore well suited for studies of protein localization (R. Owens and R. Compans, submitted for publication). HeLa T4+ cells were infected with the VV-G recombinant, and at 18 hr p.i., cells were fixed and examined using indirect immunofluorescence microscopy. The proteins expressed from VV-G were localized in a perinuclear region which coincided with the region stained by wheat germ agglutinin, a marker for the Golgi complex (Figs. 3a and b) . Recombinant VV-G-infected cells were also treated with cycloheximide to determine whether Gl and G2 glycoproteins are chased out from the Golgi region. As shown in Fig. 3 , both glycoproteins were retained in the Golgi region after a 6-hr chase with cycloheximide. The accumulation of these proteins in a localized region of the cytoplasm was much more apparent in cycloheximide-treated cells (d, e, and f) compared to the cells before treatment (c).

In order to further investigate the intracellular location of the glycoproteins, the processing of their oligosaccharide side chains was studied by treatment with endo H. After pulse labeling of VV-G-infected cells and various chase periods, proteins were immunoprecipitated and digested with endo H (Fig. 4) . The Gl synthesized from the VV-G recombinant acquired endo H resistance within a 2-hr chase period, suggesting that it contains complex-type oligosaccharide side chains. The G2 glycoprotein, on the other hand, appears to contain both high mannose-type and complex-or intermediate-type of sugar moieties. These results indicate that the Gl and G2 glycoproteins are transported from the RER to the Golgi complex where the maturation of oligosaccharides takes place.

No significant amount of surface expression of viral glycoproteins was observed on cells infected with PTV or VV-G when examined by surface immunofluorescence (Figs. 5e and f). In contrast, the vaccinia recom-binant expressing the influenza HA glycoprotein is shown as a control which exhibits typical surface protein expression (Fig. 5h) .

The lack of significant surface expresison of the PTV glycoproteins was also examined by two biochemical approaches. At 18 hr p.i., cells infected with the W-G recombinant were pulsed for 10 min with [35S]methionine, chased with medium containing excess cold methionine for 30, 60, 120, and 180 min, and the presence of proteins on cell surfaces was analyzed by surface immune precipitation. As shown in Fig. 6 , lanes fj, only faint bands were seen in the surface immunoprecipitate. No increase in the intensity of Gl or G2 proteins was seen during the chase, indicating that the proteins are not being transported to the cell surface during this time period. It was also confirmed that the ascitic fluids specific for PTV possess virus neutralizing activity, and hence have the ability to react with extracellular forms of PlV glycoproteins (data not shown). In order to determine whether the PlV glycoproteins are secreted, supernatants of W-G-infected cell cultures were examined. At 16 hr p.i., infected cells were labeled with [35S]methioninefor 6 hr, and proteins present in culture media were immunoprecipitated and analyzed by SDS-PAGE. No detectable amount of the Gl or G2 glycoproteins was found in the medium (data not shown).

Cell-surface expression was also quantitated by 1251protein A binding assays at different time points of infection. The vaccinia recombinant expressing HA was again used as a control for surface expression. As shown in Fig. 7 , HA was detectable on the cell surface at 8 hr p.i. and the amount increased until 16 hr p.i. In contrast, PTV glycoproteins were barely detectable until 16 hr p.i., and the level of maximum '251-protein A binding was significantly lower than that observed with HA. Throughout the experiment, no significant cytopathic effect was observed. The percentage of live cells in recombinant-infected cultures determined by trypan blue staining was the same as in uninfected cells.

We have constructed vaccinia virus recombinants containing a partial cDNA clone of the M genome segment of PTV, which encodes the Gl and G2 glycoproteins. Although a complete M segment clone was initially obtained, because of a mutation in the Gl glycoprotein gene and apparent inhibitory effects of the sequence preceding the Gl glycoprotein gene on bacterial growth, which interfered with attempts to repair mutations, most of the 5'sequences preceding the Gl coding sequences had to be eliminated to obtain glycoprotein expression.

This recombinant virus, W-G, efficiently produced two polypeptides that were recognized by antibody against PTV.

It has been suggested that the two glycoproteins of bunyaviruses, including PTV, are derived from a large precursor protein following proteolytic cleavage (Collett et a/., 1985; Eshita and Bishop; 1984 , lhara el a/., 1985 . Although the precursor molecule for the glycoproteins has never been identified in virus-infected cells, it was produced in an in vitro translation system using mRNA isolated from Uukuniemi virus-infected cells (Ulmanen et al., 1981) . The VV-G recombinant produced two glycoproteins similar in size to Gl and G2 from PTV-infected cells, suggesting that the cleavage between Gl and G2 occurs correctly. It was observed, however, by using tunicamycin (TM) that the unglycosylated form of G 1 -synthesized from recombinants had a slightly higher molecular weight than that from PTV-infected cells. Since only one predicted gly- At 18 hr p.i., HeLa T4+ cells infected with W-G were labeled with [35S]methionine for 10 min (lanes a and f) and chased for 30 min (lanes b and g), 60 min (lanes c and h), 120 min (lanes d and i). or 180 min (lanes e and j) in medium containing 10 mM unlabeled methionine.

Cells were lysed and reacted with anti-PTVascites fluid (lanes, a, b, c, d, and e) or treated with anti-PTV ascites fluid and then lysed (lanes f, g, h, i, and j). Following immunoprecipitation, radiolabeled polypeptides were analyzed by SDS-PAGE.

cosylation site was found in the Gl glycoprotein sequence including the extra short sequences at the 5' end in W-G (Ihara et a/., 1985) it is likely that the decrease in the size of Gl glycoproteins in the presence of TM reflects the complete inhibition of glycosylation. Also, the apparent molecular weight of the unglycosylated form of Gl glycoprotein in PTV -infected cells corresponded to the size predicted from sequence analysis (60,000 Da). The observed difference in size (2-3 kDa) between unglycosylated Gl of PTV and of W-G may, therefore, correspond to the extra 24 amino acids preceding the Gl glycoprotein, which are derived from the putative NS~ coding region. In this case, cleavage between Gl and the partial NSM sequence may not take place. Although there seems to be a consensus sequence after alanine for the potential cleavage sites in the putative precursor protein for PTV as well as Rift Valley Fever virus (RVFV) (Ihara et a/., 1985; Collett et a/., 1985) the present results indicate that an additional factor such as secondary structure of the protein may also be necessary for proper cleavage. Uncleaved precursor proteins were not detected in either PTV-or recombinant-infected cells in the presence of protease inhibitors and/or TM. Therefore, glycosylation is apparently not essential for cleavage.

The PTV glycoproteins seem to contain the necessary information for intracellular localization as well. The majority of proteins produced from the VV-G, recombinant remain intracellularly thorughout the infection period. The intracellular location of these glycoproteins was further examined by digestion of proteins with endo H. It is generally considered that the time required for the acquisition of endo H resistance corresponds to the transport time from the RER to the Golgi complex (Strous and Lodish, 1980) . The finding of endo H resistant forms of Gl and G2 glycoproteins indicates that these glycoproteins are transported from the RER to the Golgi complex. Furthermore, after 6 hr treatment with cycloheximide, both glycoproteins were still localized in the Golgi region by immunofluorescence, indicating that these proteins are retained in the Golgi complex. At late times postinfection, small amounts of proteins were detected on the cell surface by 1251-protein A binding assays but not by immunofluorescence. Since these glycoproteins were retained in the Golgi region for 6 hr in the presence of cycloheximide, any transport from the Golgi complex to the cell surface must be extremely slow if it takes place. Cell-surface expression of small amounts of glycoproteins has also been reported for other bunyaviruses (Kuismanen et a/., 1982; Madoff and Lenard, 1982; Gahmberg et a/., 1986) . However, these proteins may be present on cell-associated virions (Smith and Pifat, 1982 Schmaljohn eta/., 1987). Unlike PTV or RVFV, the HTN M genome segment lacks the 5' sequences which may encode another protein. Instead it possesses 18 amino acids following the first ATG of the M segment, and preceding the Gl glycoprotein, which may simply act as a signal peptide. These results, as well as the present observations, indicate that the glycoproteins of bunyavirus are synthesized, processed, and localized properly without a requirement for any other viral components, such as viral nucleocapsid or virus-coded nonstructural

proteins. The precise location in the glycoproteins of the signals for intracellular retention remains to be determined. A recent study of the El glycoprotein of a coronavirus (Machamer and Rose, 1987) indicated a role of hydrophobic sequences in protein retention. Deletion of one of the hydrophobic membrane spanning domains of these glycoproteins, which are normally retained at intracellular membranes, resulted in their secretion or transport to the surface. It will be of interest to carry out similar studies to determine the precise retention signal for PTV glycoproteins.

Ultrastructural study of rotavirus replication in cultured cells

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