of Heat Shock JUNJI


of Pharmaceutical Received


Sciences, March


Protein AND




1992; accepted

70 in the Rabies Virion KAWAI’


606, Kyoto,


June 25, 1992

We investigated a 73-kDa polypeptide (p73), a minor component of the rabies virion (HEP-Flury and ERA strains), accounting for as much as 1 O/O of total virion proteins. Two-dimensional gel electrophoresis and immunoblotting with the antiserum against the heat shock protein 70 (hsp70) demonstrated that p73 was identical to a constitutive type of cellular hsp70. The antiserum also detected p73/hsp70 in the purified virions of other negative-stranded RNA viruses, such as vesicular stomatitis virus (New Jersey serotype), Newcastle disease virus (Miyadera strain), and influenza A virus (PR8 strain), among which, however, the contents were variable. o 1992 Academic PWSS, h.

Purified rabies virions grown in BHK-21 cells contained, in addition to five species of major viral proteins [nucleocapsid protein (N), nonstructural phosphoprotein (M, or NS), matrix protein (M, or M), glycoprotein (G), and large protein (L)], several kinds of minor polypeptides. They were tentatively termed as pHMW (M, > 200,000), ~73, ~42, ~40, and ~351~36, respectively, according to their apparent molecular weights estimated from the relative electrophoretic mobility in SDS-polyacrylamide gel electrophoresis (PAGE) (3, 8). Among these, we have assumed that p40 and p35/p36 are isoforms of M, (the authentic form is 37 kDa; p37), because both are phosphoproteins and comigrate to the same isoelectric point as p37 in nonequilibrium pH gradient gel electrophoresis (NEPHGE) and specifically react with monoclonal and polyclonal antibodies against M, in immunoblotting and immunoprecipitation (Kawai and Sagara, unpublished data). Naito and Matsumoto (8) first demonstrated that p42 in the virion, which accounted for about 1.5% of the whole virion protein, is cellular actin, although such a possibility had been suggested for proteins of the same electrophoretic mobility found in many enveloped viruses (11). The present study focused on the identification of another minor component, ~73, which accounted for almost 1% of the whole virion proteins. The HEP-Flury and ERA strains of rabies virus, vesicular stomatitis virus (VSV; New Jersey serotype), and Newcastle disease virus (NDV; Miyadera strain) were the same ones described previously (4, 8, 2). The PR8 strain of influenza A virus was obtained from Dr. A. lshihama (National Institute of Genetics, Mishima). Anti-heat shock protein 70 (hsp70) antisera were gen’ To whom





FIG. 1. Demonstration of a minor polypeptide, ~73, in the rabies virion. The HEP-Fluty and ERA strains of rabies virus were propagated and purified as described previously (3) and were finally suspended in TBS buffer (10 mM Tris-HCI, 150 mM NaCI, pH 7.4) at a concentration of about 1 mg protein/ml. (A) Purified virions (20 fig protein) were subjected to SDS-PAGE, which was performed by using a 10% polyacrylamide slab gel and Laemmli’s discontinuous buffer system (5). Lane M, marker proteins [phosphorylase b (94 kDa), bovine serum albumin (BSA; 67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and soybean trypsin inhibitor (2 1.5 kDa)]. Lane 1, HEP strain; lane 2, ERA strain. (B) Purified HEP virions (100 pg protein) were treated with 0.25% trypsin (Difco Trypsin 1: 250; Difco Laboratories) in 1 ml of PBS(-) (137 mM NaCI, 2.7 mM KCI, 8 mM Na,HPO,, and 1.5 mM KH,PO,, pH 7.4) or mock-treated in 1 ml of PBS(-) for 5 hr at 37”, and then placed on 2 ml of 20% sucrose cushion in TBS and centrifuged at 100,000 g for 2 hr at 4”. Pellets were dissolved in 60 ~1 of SDS-PAGE sample buffer. One-third of each sample was applied to 10% SDS-PAGE. Lane 1, mock-treated control; lane 2, trypsin-treated. (C) Purified virions (40 pg protein) were dissolved with a lysis solution [2% Nonidet P-40, 5010 il-mercaptoethanol, 9.5 M urea, and 2oio Ampholine (LKB)], and were applied to 2-D gel electrophoresis, which was performed by a combination of nonequilibrium pH gradient gel electrophoresis (NEPHGE) for the first dimension and 10% SDS-PAGE for the second dimension (9). The gel was stained with 0.025% CBB. p73 is indicated by an arrowhead, and actin is indicated by arrow (A, B) or by the letter “a” (C).

be addressed. 845

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0 1992 by Academic Press. Inc. of reproduction in any form reserved.




FIG. 2. Identification of p73 as a constitutive type cellular hsp70. (A) Comparison in 2-D gels of cellular hsp70s of the heat-treated and the virus-infected BHK-21 cells. BHK-21 cells were grown on plastic plates with Eagle’s MEM supplemented with 10% Tryptose phosphate broth (Difco Laboratories) and 5% calf serum. Half of the cultures were exposed to 42” for 1.5 hr. Then, the heat-treated and mock-treated control cells were metabolically labeled with [%]methionine (20 pCi/ml) as follows: culture medium was replaced by L-methionine-free Eagle’s MEM, to which 20 &i/ml [?S]methionine (specific activity = 1050-l 150 CiimM; ARC, Inc.) was added. After incubation for 1 hr at 36”, the radiolabeled cultures were washed twice with PBS(-), and then they were lysed with a lysis solution [2% Nonidet P-40, 5% 2-mercaptoethanol, 9.5 fl/l urea, and 2% Ampholine (LKB)] and applied to the 2-D gel NEPHGE as described in Fig. 1 C. Fluorographywas performed as described elsewhere (7). 1, mock-treated control cells; 2, heat-treated cells. Arrowhead indicates hsp70, and actin is indicated by the letter “a” depicted to the left. (8) lmmunoblot analysis of p73 in the rabies virion. Purified virions (HEP and ERA strains) were prepared as described in Fig. 1. BHK-21 cells were heat-treated or infected with rabies virus (HEP strain) at an m.o.i. of 5 PFU/cell. Mock-treated cultures were also prepared as control. After dissolving the cells with an SDSPAGE sample buffer, all samples were subjected to 10% SDS-PAGE and immunoblotting, which was performed as described previously (6) by using the rabbit anti-hsp70 antiserum (a generous gift from Dr. Ohtsuka. Nagoya). Lanes 1-3, BHK-21 cell lysates: lane 1, BHK-21 cells infected with rabies vrrus (HEP strain) (10 pg protein): lane 2, normal BHK-21 cells (10 pg); lane 3, heat-shocked BHK-21 cells (10 pg). Lanes 4-6, rabies virions: lanes 4 and 5, HEP strain (20 and 10 pg protein, respectively); lane 6, ERA strain (10 pg). (C) Selective incorporation of p73ihsp70 into the rabiesvirion. Monolayer cultures of BHK-21 cells were exposed to 42” for 1.5 hr, incubated at 36” for 4 hr, and then mock-infected or infected with rabies virus at an m.o.i. of 2 PFU/cell. Control cultures which were not exposed to 42” were also treated in the same way. After incubation for 48 hr, culture fluids were harvested and subjected to the procedures for virus purification Cell lysates were obtained by lysing the cells with an SDS-

erous gifts from Dr. K. Ohtsuka (Aichi Cancer Center Research Institute, Nagoya) and Dr. K. Nagata (Chest Disease Research Institute, Kyoto University). They were prepared by immunizing rabbits with murine hsp70 which was purified either from Furth’s murine mastocytoma cells (10) or from mouse liver (Sato and Nagata, personal communication). Both antisera gave almost the same results, but antibody titer of the former was somewhat higher than that of the latter, and most experiments were performed with the former one. In addition to cellular actin, we consistently detected another minor polypeptide, ~73, in the HEP virion (Fig. 1A). After treatment with trypsin, G proteins were completely eliminated from the virion, whereas p73 as well as actin were still associated with the virion fraction, indicating that p73 is also located in the inside of the virion (Fig. 1 B). When the virion proteins were separated by two-dimensional (2-D) gel electrophoresis (9), p73 was found as a single spot (indicated by an arrowhead in Fig. 1C) locating at the site corresponding to almost the same isoelectric point as actin in the first dimension, but at a certain distance behind the spot of actin in the second dimension. Besides p73 and cellular actin, we detected in the virion preparations at least two other minor components migrating behind p73 in SDS-PAGE gel, whose nature is under investigation in our laboratory. Considering several reports on the 2-D gel analysis of cellular proteins of rodents, we supposed that the position of p73 in the 2-D gel would coincide with that of hsp70 (12). Consequently, we compared migration of p73 in the 2-D gel with that of cellular hsp70s in the heat-treated and untreated control BHK-21 cells (Fig. 2A). Judging from their relative mobilities in 2-D gels, we assumed that the position of p73 corresponded to or very close to that of one of hsp70s, the one which is known to be a constitutive type (73 kDa) of hsp70. Another hsp70 of faster mobility (70-kDa hsp70), which was heat-inducible and could not be detected in normal untreated BHK-21 cells, did not seem to correspond to ~73. Furthermore, when nonlabeled purified virions and the lysate of [35S]methionine-labeled heatshocked BHK-21 cells were mixed and applied to 2-D gel NEPHGE, the Coomassie brilliant blue (CBB)

PAGE sample buffer. Virions (20 pg protein) and cell lysates (20 pg protein) were subjected to 10% SDS-PAGE and immunoblotting with the anti-hsp70 antiserum. Lane 1, rabies virions produced by the preheated cells; lane 2, the lysate of preheated infected cells. A band seen below that of p73/hsp70 (lane 2) is of the inducible type hsp70. The lanes for the mock-infected cultures were omitted from Fig. 2C, since the results were the same as seen in Fig. 2B.



FIG. 3. Virion-associated hsp70 of other negative-stranded RNA viruses. (A) Purified VS virions were prepared as described previously (3) and finally suspended in TBS buffer at a concentration of about 1 mg protein/ml and subjected to 10% SDS-PAGE and immunoblotting as described in Fig. 2B. Lanes M and 1: CBB-stained SDS-PAGE gel [lane M, marker proteins (phosphorylase b, bovine serum albumin, ovalbumin. chymotrypsinogen A); lane 1, VSV (20 pg protein)]. Lanes 2 and 3: immunoblotting [lane 2. VSV (10 pg); lane 3, rabies vrrus (HEP strain) (10 rg)]. Letters depicted to the right of lane 1 indicate VS vrrion proteins. Arrowheads indicate ~73. (B) Virion samples of NDV and influenza virus were prepared as follows: 1 OOOfold diluted virus stocks were inoculated into the chorioallantoic cavity of g-day-old chrcken eggs. After incubation at 37” for 48 hr, virus samples were collected and clarified by low-speed centrifugation (2000 g) for 30 min, which was followed by ultracentrifugatron at 68,000 g for 90 min at 4”. Virus pellets were resuspended in TSE buffer (0.01 MTris-HCI, 0.15 M NaCI, and 1 mlVI EDTA, pH 7.4) and subjected to two cycles of density gradient centrifugation. The first was isopycnic centrifugation through a lo-45% potasium tartrate gradient at 68,000 g for 4 hr. Viruses recovered from a single band were subjected to the second cycle of ultracentrifugation through another 15-40% potasium tartrate gradient at 68,000 g for 4 hr (NDV) or through a 1 O-40% sucrose gradient in TSE buffer at 52,000 g for 80 min (influenza A virus). Virions recovered from the second cycle density gradient centrifugation were sedimented again at 68,000 g for 1 hr, and were resuspended in TBS buffer at a final concentration of about 1 mg protein/ml. Lysates of the normal and the heat-shocked (exposed to 43” for 1 hr) secondary cultures of chicken embryo frbroblasts (CEF) were prepared by lysing the cells with an SDS-PAGE sample buffer. All samples were applied to two sets of 10% SDS-PAGE gels. After electrophoresis, they were processed for CBB-staining or the immunoblotting as described in Fig. 26. Lanes 1 and 2: CBB staining [lane 1, influenza A vrrus (10 pg); lane 2, NDV (10 fig)]. Lanes 3-6: immunoblotting [lane 3, normal CEF (20 Hg); lane 4, influenza virus (20 pg); lane 5, NDV (20 pg); lane 6, heat-treated CEF (20 fig)]. Letters depicted to the right and left of the lanes 1 and 2 indicate virion proteins of influenza virus and NDV, respectrvely. Arrowheads indicate ~73, and an arrow actin.

stained spot of p73 exactly corresponded to the spot of 73-kDa hsp70 as visualized by fluorographic procedures (data not shown). This assumption was reexamined by the immunoblot method using the specific antihsp70 antisera, which recognized the two species of hsp70s (70 and 73 kDa) of BHK-21 cells (Fig. 2B, lane 3). As shown in Fig. 2B (lanes 4, 5, and 6) the antisera detected only a single type of hsp70 in the rabies vi-


rion, whose electrophoretic mobility corresponded to that of ~73. Another hsp70 (70-kDa hsp70) was not detected in the virion even though increased amounts of virion proteins were applied to the immunoblots (Fig. 2B, lane 5). Furthermore, 70-kDa hsp70 could not be detected in the HEP virus-infected cells (Fig. 2B, lane 1). Since the relative content of p73/hsp70 in the virion was similar to that in the cells as judged from immunoblot assays (Fig. 2B), and most cellular proteins were excluded from the virions, we assume that p73/hsp70 was, although not enriched, selectively enclosed into the virion. Furthermore, only the constitutive type ~731 hsp70 was incorporated into the virion even when the virus was grown in the preheated host cells (Fig. 2C, lane l), in which similar amounts of p73/hsp70 and the inducible 70-kDa hsp70 were produced (Fig. 2C, lane 2). The preheating of host cells did not affect significantly the yield of progeny virus (data not shown). As to the ERA strain of rabies virus, the band of virion-associated p73 could not be seen in the CBBstained one-dimensional SDS-PAGE gels due to an overlapping broad band of G protein (Fig. IA, lane 2). Two-D gel NEPHGE, however, separated individual spots of p73 and G protein of the ERA virion according to their different isoelectric points (data not shown). lmmunoblot analysis also detected a single distinct band at the same position as p73 of the HEP virus, and its relative content was almost the same as that in the HEP virions (Fig. 2B, lane 6). From these results, we conclude that only a constitutive type (p73), but not inducible type (70 kDa), of cellular hsp70s was selectively incorporated into the rabies virion as a regular component probably in specific or functional association with certain virion component(s). We next examined whether incorporation of hsp70 into the virion is a more common phenomenon occurring in many types of viruses. lmmunoblot analysis demonstrated that VS virions (New Jersey) grown in BHK-21 cells also contained p73/hsp70 (Fig. 3A), although the amount was much less when compared with the rabies virion. Next, we examined other negative-stranded RNA viruses, such as NDV (paramyxovirus) and influenza A virus (orthomyxovirus). Before doing this, we first checked whether the anti-hsp70 antiserum could recognize avian hsp70s by the immunoblot method, because these viruses were propagated in the embryonated chicken eggs. As shown in Fig. 3B (lane 3) the antiserum recognized hsp70 of chick embryo fibroblasts (CEF) and detected the increased synthesis of hsp70 in the heat-treated CEF (Fig. 3B, lane 6). Although a large amount of HN protein (about 75 kDa) of NDVvirions concealed a supposed thin band of



hsp70 in a CBB-stained SDS-PAGE gel, the immunoblotting with the antiserum detected hsp70 in the virion, but the band was pressed frontward by a huge broad band of HN (Fig. 3B, lanes 2 and 5). Purified virions of influenza A virus also contained hsp70; in a CBB-stained SDS-PAGE gel, a faint band was visible at the corresponding site of p73/hsp70 (Fig. 3B, lane l), and its relative content was much less than in NDV as demonstrated by immunoblotting (Fig. 3B, lane 3). The anti-hsp70 antiserum used in this study was prepared against the murine hsp70, but was shown to be effective for detecting the avian hsp70. By using this, we identified one of minor virion components, ~73, as cellular hsp70, which was also shown to be a regular minor component of all negative-stranded (segmented and nonsegmented) RNA viruses examined in this study (i.e., rabies virus, VSV, NDV and influenza A virus). Relative contents of p73/hsp70 in the virion seemed to be quite variable depending on the species of the virus; for instance, the content was quite different between VSV and rabies virions grown in BHK-21 cells. We cannot tell, however, whether the antiserum binds to the hamster and avian hsp70s at the same efficiency. Accordingly, we could not compare quantitatively the relative content of hsp70 in the rabies and NDV virions, which were grown in BHK-21 cells and in chicken eggs, respectively. Avian hsp70 displayed approximately the same electrophoretic mobility as p73/ hsp70 of BHK-21 cells in SDS-PAGE gels (data not shown). The chicken hsp70 may consist of at least two species (I). However, we could not distinguish them in one-dimensional SDS-PAGE as well as in 2-D NEPHGE, since they migrated at a similar rate in the

gels. Accordingly, it is uncertain so far whether the antiserum recognized either one or both of them, so we could not determine which type of the two was incorporated into the virions grown in chicken eggs.

ACKNOWLEDGMENTS We are most grateful to Dr. K. Otsuka (Aichi Cancer Center Research Institute, Nagoya) as well as to Dr. K. Nagata and Mr. M. Sato (Chest Disease Research Institute, Kyoto University) for their kind supply of antisera against hsp70. Gratitude is also to Miss I. Kodera for her help in manuscript preparation.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11.


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Identification of heat shock protein 70 in the rabies virion.

We investigated a 73-kDa polypeptide (p73), a minor component of the rabies virion (HEP-Flury and ERA strains), accounting for as much as 1% of total ...
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