LD-652 Cells by Vaccinia Virus

VIROLOGY 248, 74±82 (1998) ARTICLE NO. VY989241

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The Non-Permissive Infection of Insect (Gypsy Moth) LD-652 Cells by Vaccinia Virus

Yi Li, Shala Yuan, and Richard W. Moyer1

Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida 32010±0266 Received March 12, 1998; accepted May 19, 1998

The members of Poxviridae family are among the most complex of animal viruses and subfamily members infect both vertebrate (Chordopoxvirinae) and invertebrate (Entomopoxvirinae) hosts, respectively. Vaccinia virus (VV) is the most commonly studied vertebrate virus and the entomopoxvirus of Amsacta moorei (AmEPV) is the prototypic insect virus. AmEPV, while not able to productively infect vertebrate cells, does enter vertebrate cells and expresses early genes after which the infection aborts although the cells survive (Y. Li, R. L. Hall, and R. W. Moyer. J.Virol. 71(12), 95579562, 1997). We show here that a recombinant VV, containing the lacZ gene regulated by the cowpox virus A-type inclusion (ATI) late promoter, likewise does not productively infect insect cells. Our results suggest that the recombinant VV enters insect cells, host protein synthesis is inhibited, early gene expression is normal, and viral DNA replication occurs as does late protein synthesis. However, little if any proteolytic processing of late viral proteins, typical of morphogenesis, is observed. Electron micrographs of infected cells suggest that while cytoplasmic virosomes (factories) are formed, there is little indication of further morphogenesis or any formation of mature virions. Therefore, while both orthopoxviruses and entomopoxviruses fail to replicate in heterologous hosts, the nature of abortive infections is quite different. © 1998 Academic Press

INTRODUCTION virion morphology, a large, linear, double-stranded DNA genome (190 kb for VV, 225 kb for AmEPV), and common The Poxviridae family, comprises a large number of sequence motifs which are associated with promoter complex DNA viruses which are characterized by a cy- elements and nonspliced transcripts. There are also toplasmic site of replication. Poxviruses infect a variety of some significant differences. For example, the genomes vertebrate and invertebrate hosts and are subdivided of the two viruses differ widely in G ϩ C content (37% for into two subfamilies, the Chordopoxvirinae (poxviruses VV, but only 18% for AmEPV), length of the replication of vertebrates) and the Entomopoxvirinae (poxviruses of cycle, optimal growth temperature (37°C for VV, 26±28°C insects). for AmEPV), and most obviously host range (Moyer, 1994; Vaccinia virus (VV) is the most extensively studied Arif, 1995). The limited serology available suggests that vertebrate poxvirus. The Entomopoxvirinae (EPV) were there is little immunological relationship between the only recently discovered (Vago, 1963) but are now known two viruses. to have a worldwide distribution and to productively A key consequence of the poxvirus replicative strategy infect primarily the Orthoptera, Diptera, Coleoptera and is that upon entry into cells, early viral genes are tran- Lepidoptera orders of insects (Moyer, 1994; Arif, 1995). scribed by enzymes packaged within the infecting virion The lepidopteran entomopoxvirus isolated from Amsacta core that are derived from viral proteins synthesized and moorei (AmEPV) is the first insect poxvirus to be adapted to grow in cultured insect cells (Granados, 1981; Good- packaged from the infected cells of the previous growth win et al., 1990; Dall et al., 1993) and is therefore one of cycle. Following early gene expression and a second the best studied. More recently, a second EPV (Fernon et step of uncoating, viral DNA replication ensues leading al., 1995) from Heliothis armigera has also been adapted to formation of cytoplasmic electron-dense virosomes or to cell culture. ``factories'' consisting of viral DNA. DNA replication is a While VV and AmEPV share an overall biological strat- requirement for the initiation of intermediate and late egy and have some properties in common, there are also viral gene expression. In the case of VV, a series of some striking differences. Similarities include a similar proteolytic events heralds the onset of morphogenesis followed by virion assembly and concomitant packaging of the early transcription apparatus necessary to initiate the next round of infection. 1 To whom correspondence and reprint requests should be addressed Late in AmEPV infection, some virions typically be- at Department of Molecular Genetics and Microbiology, College of Med- icine, University of Florida, PO Box 100266, JHMHC, Gainesville, FL 32010± come embedded within a protein matrix forming large 0266. Fax: (352) 846 2042. E-mail: [email protected]. cytoplasmic occlusion bodies or spheroids which con-

0042-6822/98 $25.00 Copyright © 1998 by Academic Press 74 All rights of reproduction in any form reserved. VACCINIA VIRUS INFECTION OF INSECT CELLS 75 sist primarily of a single protein, spheroidin (Bilimoria infected vertebrate cells express only early viral genes and Arif, 1979; Langridge and Roberts, 1982; Hall and and that there is neither AmEPV viral DNA synthesis nor Moyer, 1991; Banville et al., 1992). Spheroidin can com- expression of AmEPV late genes (Li et al., 1997). We prise 30±40% of the protein of the infected insect cell. began our studies of VV infection of insect cells by first Spheroidin is functionally similar to the highly expressed analyzing infected cells for the expression of ␤-galacto- A-type inclusion (ATI) protein of cowpox virus (CPV) (Pa- sidase derived from either AmEPV or VV recombinant tel et al., 1986; Funahashi et al., 1988) which can seques- viruses where the lacZ gene is regulated by the CPV ATI ter CPV within paracrystalline inclusions. This gene is gene promoter. Expression of ␤-galactosidase was defective in VV (Amegadzie et al., 1992). Structurally, the readily observed in both cases (Figs. 1C and 1D). No AmEPV spheroidin and CPV ATI gene promoters appear ␤-galactosidase expression was seen in either mock- to be quite similar, being AT rich and containing the infected cells or cells infected with wt AmEPV (Figs. 1A ``TAAATG'' motif characteristic of a wide variety of verte- and 1B). The proportion of stained cells in recombinant brate poxvirus late promoters (Davison and Moss, 1989). VV-infected cells was dose dependent but the relative Despite these similarities, there is very little spheroidin efficiency noted for infection of insect cells at an m.o.i. of expression when the spheroidin gene is inserted into the 10 (as determined in vertebrate cells) was only 15±20% genome of either VV or CPV (Hall et al., 1996; Li et al., that of the vertebrate cells. We have found that a VV 1998) suggesting that both the vertebrate and inverte- multiplicity of 100 is needed to infect virtually all of the brate viruses each have unique regulatory features. insect cells. Consistent with that prediction, we have recently The levels of ␤-galactosidase expressed by the VV found that AmEPV infection of vertebrate cells leads to recombinants was also quantified and compared to an abortive infection. AmEPV enters vertebrate cells and AmEPV lacZ recombinant viruses. Insect cells were in- early, but not late genes are expressed (Li et al., 1997). fected at an m.o.i. of 10 with AmEPV or 100 with VV Similarly, when VV was used to infect BTI±EAA insect recombinant viruses each containing lacZ under control cells, some viral protein synthesis was detected 10 h of the CPV ATI promoter and allowed to adsorb for 2 h, after infection by enzyme-linked immunoadsorbent assay after which the media was removed and replaced with (ELISA) using vaccinia virus antisera, indicating viral fresh media with or without cytosine 1-␤-D-arabino- entry into the insect cells. The protein(s) detected in furanoside (AraC). At various times thereafter, cells were these very early studies were assumed to represent VV harvested and the levels of ␤-galactosidase evaluated. structural proteins (Langridge, 1983a,b). VV DNA replica- As shown in Fig. 2, in the absence of AraC, ␤-galactosi- tion was not detected and the infection was assumed to dase activity can be detected from either recombinant be abortive. The inability of EPVs to grow despite entry virus by 12 h after infection with levels increasing until into vertebrate cells and, similarly, the inability of vac- 48 h postinfection. The absolute level of AmEPV-medi- cinia virus to grow in insect cells further emphasizes the ated ␤-galactosidase appears to be about twice that unique features of the two subfamilies of poxviruses. from the VV recombinant, however, this difference may We have more carefully examined the infection of still in part reflect incomplete infection of all the insect insect cells by VV. We show that VV viral DNA replication cells by VV. The ␤-galactosidase expression is mediated occurs in VV-infected insect cells as does late gene by late promoter activity as expected because in both expression as indicated by ␤-galactosidase expression cases ␤-galactosidase expression was completely inhib- from a lacZ reporter gene regulated by the CPV ATI late ited by the addition of the AraC. gene promoter. Analysis of late viral proteins by pulse± chase experiments gives no evidence of the proteolytic Analysis of viral DNA synthesis processing of late proteins typical of viral morphogenesis necessary for a productive infection. While numerous The data of Figs. 1 and 2 show that VV infection of virosomes or ``factories'' were observed in VV-infected insect cells results in late promoter regulated gene ex- insect cells, few assembly intermediates or virions were pression. During a VV infection, viral DNA replication is a noted, suggesting that impairment of morphogenesis ul- prerequisite for late viral gene expression and hence timately leads to an abortive infection. unlike AmEPV-infected vertebrate cells, where the infection arrests prior to viral DNA synthesis, we would expect RESULTS VV viral DNA synthesis to occur in the infected insect cells. Therefore, VV-infected insect cells were examined ␤-galactosidase expression in vaccinia recombinant for viral DNA synthesis (Fig. 3). As expected, VV viral infected cells DNA synthesis is readily observed, first detectable by Both AmEPV infection of vertebrate cells and VV infec- 12 h postinfection (Fig. 3B) consistent with the kinetics tion of insect cells are generally accepted to be nonpro- observed for expression of late promoter regulated ␤-ga- ductive. We have previously demonstrated that AmEPV- lactosidase. The time of induction of DNA synthesis was 76 LI, YUAN, AND MOYER

FIG. 1. X-Gal staining of LD-652 cells infected with recombinant AmEPV ATI-LacZ or the recombinant VV ATI-LacZ. Cells were stained with X-Gal 48 h postinfection. (A) Mock-infected cells. (B) Wild-type AmEPV-infected cells at an m.o.i. of 10. (C) Recombinant AmEPV ATI-LacZ-infected cells at an m.o.i. of 10. (D) Recombinant VV ATI-LacZ-infected cells at an m.o.i. of 100. identical to that observed for AmEPV-infected insect cells, but the levels of AmEPV DNA synthesis were con- sistently higher despite the fact that the multiplicity of VV used assured that virtually all the insect cells were infected. It should be noted that the lacZ gene in VV is located within the TK gene, rendering the virus TK negative. The AmEPV contains lacZ inserted within the spheroidin gene. It is unlikely, however, that the lower level of VV DNA synthesis is related to TK inactivation because the LD cells used in these experiments contain a functional TK gene (Hall et al., 1996; Li et al., 1997).

VV-infected insect cells fail to produce infectious virus Since both viral DNA synthesis and late gene expression FIG. 2. Comparison of AmEPV- and VV- recombinant mediated-␤± occur in VV-infected insect cells, we next determined galactosidase expression in LD-652 cells. Cells were infected with AmEPV whether progeny virus was produced. Insect cells were ATI-lacZ at an m.o.i. of 10 pfu/cell or VV ATI-lacZ at an m.o.i. of 100 pfu/cell. infected with either VV ATIlacZ or AmEPV ATIlacZ.Two After2hofincubation, the virus containing media was removed and hours after addition of virus, the inoculum was removed, replaced with fresh medium. Infected cells were harvested at the times fresh media added and the cells further incubated at 26°. indicated and extracts were prepared and assayed for ␤-galactosidase activity. Each point represents the average of three independent assays of The infected cells were harvested at various times, the cells AmEPV ATI-lacZ in the presence of AraC (Œ) or absence of AraC (f), and disrupted to release any intracellular virus and titered. As of VV ATI-lacZ-infected cells in the presence of AraC (ࡗ) or in the absence shown in Fig. 4, the production of AmEPV is readily detect- of AraC (●). O.D. 420, optical density at 420. VACCINIA VIRUS INFECTION OF INSECT CELLS 77

infection. Therefore, we next examined the patterns of newly labeled viral proteins in VV-infected LD-652 and CV-1 cells. Infected cells were pulse-labeled as described (Winter et al., 1995) in Materials and Methods at periods throughout the infection and the labeled proteins then analyzed by SDS polyacrylamide gel electrophoresis. The overall patterns of viral protein synthesis in VV ATI-lacZ-infected insect and CV-1 cells are similar (Fig. 6) and typical of the patterns expected for late viral proteins. However, there also appears to be generally less viral protein synthesis in the VV- FIG. 3. Viral DNA synthesis in infected LD-652 insect cells. DNA synthesis was examined in AmEPV ATI-lacZ- infected (m.o.i. ϭ 10) cells infected insect cells. The lower levels of late VV pro- (A) and VV ATI-lacZ-infected (m.o.i. ϭ 100) cells (B). Cells were infected, tein synthesis in the infected insect cells may reflect in the virus was removed after2hofabsorption, and fresh medium was part the lower growth temperature (28°C) necessary added. Infected cells were harvested at the time indicated and viral for the insect cells. Host protein synthesis is ultimately DNA was extracted. DNA synthesis was then measured by dot blot inhibited in both cases which provides an indication analysis using a radiolabeled lacZ DNA probe as described in Mate- rials and Methods. U, DNA from mock-infected cells. that VV has infected all the insect cells. Proteolytic processing of late VV proteins is indicative of viral morphogenesis. These proteolytic cleavages are able by 24 h postinfection and increases to about 72 h post readily observed in vertebrate poxvirus-infected cells infection. No production of infectious VV was observed when radiolabeled newly synthesized proteins are al- despite the fact that both viral DNA and late protein syn- lowed to incubate further after removal of the radiolabel. thesis are observed, suggesting a defect in the assembly of Particularly noticeable, under such experimental condi- infectious virus. Attempts to rescue VV infection of insect tions is the processing of two major structural proteins of cells by co-infection with AmEPV were unsuccessful (data VV, p4a and p4b, which are cleaved to mature forms of 63 not shown). and 60 kDa, respectively (Moss et al., 1969; Katz and Moss, 1970a,b; Rosel and Moss, 1985; Van Meir and Electron microscopic examination of VV-infected Wittek, 1988; VanSlyke and Hruby, 1990). Therefore, to insect cells evaluate protein processing of newly labeled proteins, the labeled infected cells were further incubated in non- Electron microscopic analysis of infected cells was radioactive media. Proteolytic processing of VV structural undertaken to determine whether VV-infected insect cells are capable of forming any structures consistent with viral assembly. Insect cells were infected with either recombinant VV or AmEPV and at 48 h postinfection, cells were prepared for examination by electron microscopy (Fig. 5). In VV-infected insect cells, cytoplasmic virosomes or ``factories'' were readily observed (Fig. 5A) as on occasion were viral crescent- like structures (Fig. 5B), but no spherical immature particles nor mature virions were seen. Viral crescents are one of the earliest structures associated with viral morphogenesis. By contrast, mature virions were readily visible in AmEPV infected cells (Figs. 5C and 5D). Characteristics of mature AmEPV virions include the formation of a well-differentiated core (Fig. 5D). Since the AmEPV ATIlacZ recombinant contains the lacZ gene inserted within the spheroidin gene, the resultant virus is spheroidin negative and no occlusion FIG. 4. Titration of infectious VV ATI-lacZ in LD-652 cells. LD-652 body-related structures are observed. cells were infected with recombinant VV ATI-lacZ at an m.o.i. of 100 or AmEPV ATI-lacZ at an m.o.i. of 10. After2hofincubation, cells were Viral protein synthesis and processing in VV-infected washed twice with serum-free medium and then fresh medium was insect cells added. Cells and supernatants were harvested together at the times indicated and sonicated. Cellular debris was removed by centrifuga- It would appear that the block in the nonpermissive tion. The clarified AmEPV (f)- and VV (●)-infected supernates were VV infection of insect cells involves the late stages of then titered on LD-652 cells or CV-1 cells, respectively. 78 LI, YUAN, AND MOYER

FIG. 5. Transmission electron microscopy of VV- or AmEPV-infected insect cells. VV ATI-lacZ-infected (A and B) and AmEPV ATI-lacZ-infected (C and D) LD-652 cells at 48 h postinfection. (A) Arrowhead indicates newly replicated DNA of ``virosomes'' or ``factories.'' (B) Arrowhead shows bilayer membrane ``crescent'' surrounding immature virions. (C) Arrowhead indicates mature AmEPV virion with a well-developed core. (D) Mature AmEPV virions at somewhat higher magnification. proteins was readily visible in VV infected control CV-1 ences from the data shown, i.e., Figs. 6 and 7 where cells at 8 and 24 h after labeling of the proteins (Fig. 7A) proteolysis is readily observed in infected CV-1 cells but but not in infected LD-652 cells (Fig. 7B). There is again not infected insect cells. It is also interesting to note that the question as to whether the failure to observe VV unlike VV late proteins, the processing of AmEPV late protein proteolysis in infected cells is related to the lower viral proteins during a normal infection of LD-652 cells temperature (28°g) of infection. We have addressed this has not been observed (Winter et al., 1995). The apparent question by analyzing VV protein proteolysis in both absence of the processing of late AmEPV proteins re- VV-infected LD-652 and CV-1 cells at 32°C, an interme- flects a fundamental difference between the vertebrate diate temperature for both cell lines. We found no differ- and insect poxviruses (Winter et al., 1995). VACCINIA VIRUS INFECTION OF INSECT CELLS 79

DISCUSSION We have previously shown that while AmEPV does not productively infect vertebrate cells, the virus does enter vertebrate cells and limited (early) viral gene expression occurs. More specifically, expression of a lacZ reporter gene was observed in recombinant AmEPV-infected vertebrate cells if the lacZ gene was under control of either of two entomopoxvius early gene promoters but not when lacZ was regulated by either of two viral late promoters (Li et al., 1997). Furthermore, no viral DNA synthesis was observed in AmEPV-infected vertebrate cells, and no reporter gene expression was observed if the reporter gene was under control of late promoters (Li et al., 1997). Therefore, the infection aborts after early gene expression. In contrast, the infection of insect cells by VV, while FIG. 7. Late protein processing in VV ATI-lacZ-infected CV-1 and also abortive, generates quite a different nonpermissive insect cells. CV-1 cells (A) or LD-652 cells (B) were infected with VV phenotype in that the block appears to be quite late in ATI-lacZ at multiplicities of 10 and 100, respectively. Late proteins were the infectious cycle. Expression of lacZ under control of pulse labeled at 12 h postinfection as described in Fig. 6 and Materials either a strong late vertebrate poxvirus (ATI) or AmEPV and Methods. The label was then removed, and the infected cells (spheroidin) promoter is readily apparent (Figs. 1 and 2). further incubated for the periods of time indicated in the figure. The samples were then harvested and analyzed by SDS-polyacrylamide gel VV viral DNA synthesis was also observed within in- electrophoresis. Molecular weight size standards are indicated on the fected insect cells (Fig. 3) but no virions are formed (Figs. left. U, mock-infected cells. In (A), the open arrows indicate the pro- 4 and 5). Although early studies (Langridge, 1983a,b) cessed, mature VV late 4a and 4b proteins. In (B), the closed arrow showed some indications of viral protein synthesis in shows the position of ␤-galactosidase. VV-infected BTI-EAA insect cells early after infection, there was no evidence of viral DNA synthesis (Lang- ridge, 1983a,b). Poxvirus gene expression is primarily regulated at the level of transcription. During an infection, VV genes are transcribed via a cascade mechanism comprising three temporally distinct classes of mRNAs: early, intermediate, and late. Early genes are transcribed by enzymes packaged within the virion. A subset of early proteins are then able to act as transcription factors required for the synthesis of intermediate genes and a subset of intermediate genes serve as late gene transcription factors. Both intermediate and late gene expression also depend on viral DNA replication. Since we observe both viral DNA replication (Fig. 3) and late promoter mediated reporter gene expression (Figs. 1, and 2) as well as viral late proteins (Figs. 6 and 7), it is safe to assume that both early and intermediate VV gene expression is relatively normal. Labeling of newly synthesized VV proteins within infected insect cells (Fig. 6), clearly suggests late genes are expressed, however, no processing of late proteins is observed (Fig. 7). Electron microscopic examination (Fig. 5) of infected cells suggests that few if any virions are FIG. 6. Analysis of newly labeled proteins in VV-infected insect cells formed. DNA containing Virosomes (factories) are readily and vertebrate cells. VV ATI-lacZ-infected CV-1 (A) and LD-652 (B) cells. observed, but no morphological structures normal asso- U, mock-infected cells and the times after infection (h) for the initiation of the pulse-labeling of proteins are indicated. The arrow indicates the ciated with viral assembly are observed with the excep- position of ␤-galactosidase. Molecular weight size standards are indi- tion of a few membrane ``crescent,'' structures thought to cated on the left. be among the very earliest of intermediates associated 80 LI, YUAN, AND MOYER with viral morphogenesis (Fig. 5). We have attempted to strain (Condit and Motyczka, 1981) was grown at 37°C in complement VV infections of insect cells by coinfection CV-1 cells, maintained in Eagle's minimal essential me- with AmEPV to no avail. dia (MEM) with Earle's salts supplemented with 5% FBS. What specific aspect of VV infection of insect cells is Rat TK (Ϫ) cells were maintained in MEM media con- defective? There are at least two possibilities. The first is taining BUdR (100 ␮g/ml). that the proteinase(s) responsible for processing of late VV proteins in vertebrate cells is absent in insect cells. Construction and selection of recombinant viruses The implication of this hypothesis is that the protein- pSPH-ATIlacZ and pVATIlacZ were constructed and ase(s) is cellular in origin. A second possibility relates recombinants were selected as described previously directly to transcription. While VV protein synthesis ap- (Hall et al., 1996; Li et al., 1997, 1998). All inserts were pears relatively normal in infected insect cells, we do not within the TK gene of the respective viruses or for know that all VV promoters are recognized and utilized. It AmEPV, also within spheroidin (Hall et al., 1996). The has been shown that a components of both intermediate stable vaccinia recombinants were selected on rat TK (Rosales et al., 1994a,b) and late VV transcription factors (Ϫ) cells in the presence of BUdR using standard tech- (Wright et al., 1998) are cellular in origin. It is possible niques (Smith and Moss, 1984). Virus titer was estimated that the corresponding transcription factor subunits con- by plaque assay. tributed by the insect cells are either absent, nonfunc- tional, or only partially able to function with the transcrip- Assay of ␤-galactosidase, pulse-labeling and pulse± tional apparatus of the vertebrate VV. chase of proteins in VV-infected CV-1 and LD 652 Although the metabolic blocks in the two heterologous cells, and SDS±PAGE sytems is clearly different, the fact that both viruses enter their respective nonpermissive host cells suggests that IPLB-LD-652 cells (2 ϫ 105 cell/well) or CV-1 cells (4 ϫ the cellular receptors that mediate binding and entry 105) in 12-well plates were infected with recombinant have been relatively conserved. This is an interesting AmEPV at an m.o.i. of 10 pfu/cell or VV at an m.o.i. of 10 observation considering that in general, unlike VV, ento- or 100 PFU/cell for CV-1 and IPLB-LD 652 cells, respec- mopoxviruses, do not generally grow in cultured cells. tively, for 2 h, unless stated otherwise. After 2 h, the Indeed, AmEPV is one of the few that has been adapted inoculum was removed, the cells were rinsed with se- to replicate in cultured insect cells (Granados, 1981; rum-free TE media or MEM media, and 1 ml of fresh Goodwin et al., 1990; Hukuhara et al., 1990; Marlow et al., medium with or without cytosine 1-␤-D-arabino-furano- 1993; Fernon et al., 1995). side (AraC) (200 ug/ml) was added to each well. At One of the most intriguing aspects of the AmEPV appropriate times postinfection, the infected cells were abortive infection of vertebrate cells is that the limitation harvested by scraping into the media and freezing at of early gene expression can be reversed in part by Ϫ70° C until assayed. The ␤-galactosidase (␤-gal) activ- coinfection with VV (Li et al., 1997). Mixed infection of ity assays were performed using O-nitrophenyl-␤-D-ga- vertebrate cells by both VV and AmEPV allows expres- lactopyranoside (ONPG) as the substrate as described sion of reporter genes within the AmEPV genome regu- previously (Miller, 1972). ␤-gal activity was measured at lated by either the ATI or spheroidin late promoters. 420 nm of absorbance and normalized for OD420 per mg Therefore, VV encodes a gene or genes which facilitate of total protein (Bradford, 1976). The staining of ␤-galac- further expression of the AmEPV genome in vertebrate tosidase in situ was performed 48 h after infection. The cells. Whether this gene(s) is related to direct facilitation infected cells were washed with PBS, fixed with a solu- of transcription or perhaps to the uncoating of the virus tion of 2% formaldehyde plus 0.2% glutaraldehyde pre- particle are but two of the most obvious possibilities pared in PBS and stained with a reaction mixture of which are currently under investigation. 1 mg/ml 5-bromo-4-chloro-3-indolyl-␤-D-galactosidase (X-gal), 5 mM potassium ferricyanide, 5 mM potassium MATERIALS AND METHODS ferrocyanide, and 2 mM magnesium chloride. Pulse-labeling of newly synthesized proteins for 1-h Cells and virus periods in either AmEPV- or VV-infected LD-652 insect AmEPV (Hall and Moyer, 1991) was grown in IPLB-LD- cells was performed as previously described (Winter et 652 cells (Goodwin et al., 1990) maintained at 28°C in a al., 1995). LD-652 cells (2 ϫ 105 cells/well) or CV-1 cells 1:1 mixed medium (TE medium) of TC-100 media (Gibco, (4 ϫ 105 cells/well) in 12-well plates were infected with Gaithersburg, MD) and EX-CELL 401 media (JRH Bio- VV ATI-lacZ at an m.o.i. of 100 or 10 pfu/cell, respectively, sciences, Lenexa, KS), supplemented with 10% fetal bo- and the virus adsorbed for 2 h. The inoculum was then vine serum (FBS). Isolation of a thymidine kinase (TK) removed, the cells rinsed with serum-free media and negative TK (Ϫ) IPLB-LD-652 cell line, C11.3 has been then incubated in regular media. Before labeling, the described earlier (Li et al., 1997). Vaccinia virus (VV) WR cells of individual wells were washed with 1 ml of me- VACCINIA VIRUS INFECTION OF INSECT CELLS 81 thionine-free EX-CELL 401 (JRH biosciences) or 1 ml of Dall, D., Sriskantha, A., Vera, A., Lai-Fook, J., and Symonds, T. (1993). A methionine-free minimum essential Eagle's medium gene encoding a highly expressed spindle body protein of Heliothis (Sigma) and incubated in these methionine-free media armigera entomopoxvirus. J. Gen. Virol. 74, 1811±1818. Davison, A. J., and Moss, B. (1989). Structure of vaccinia virus late for 45 min to deplete amino acid pools. The cells were promoters. J. Mol. Biol. 210, 771±784. then incubated in 0.4 ml methionine-free EX-CELL me- Fernon, C. A., Vera, A. P., Crnov, R., Lai-Fook, J., Osborne, R. J., and Dall, dium or minimum essential Eagle's medium containing D. J. (1995). Replication of Heliothis armigera entomopoxvirus in vitro. 60 ␮Ci/ml Trans 35S-label (ICN Pharmaceuticals, Inc., J. Invertebr. Pathol. 66, 216±223. Irvine, CA) and incubated for 1 h. The cells were then Funahashi, S., Sato, T., and Shida, H. (1988). Cloning and characteriza- washed with PBS to remove unincorporated label and tion of the gene encoding the major protein of the A-type inclusion body of cowpox virus. J. Gen. Virol. 69, 35±47. lysates prepared for analysis via protein electrophoresis. Goodwin, R. H., Adams, J. R., and Shapiro, M. (1990). Replication of the To evaluate proteolytic processing of late proteins, the entomopoxvirus from Amsacta moorei in serum-free cultures of a radioactive medium was removed after the 1-h labeling gypsy moth cell line. J. Invertebr. Pathol. 56, 190±205. period from the cells and replaced with regular media. Granados, R. R. (1981). ``Pathogenesis of Invertebrate Microbial Diseas- The cells were then further incubated for additional pe- es'' (Davidson, E.W., Ed.), pp. 101±126. Allanheld, Osmun & Co., riods of time as indicated. Harvest of samples and anal- Totowa, N.J. Hall, R. L., Li, Y., Feller, J. A., and Moyer, R. W. (1996). 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