Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines

Virus Research 93 (2003) 13 /23 www.elsevier.com/locate/virusres

Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines Md. Masmudur Rahman, Karumathil P. Gopinathan * Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India Received 8 November 2002; received in revised form 9 January 2003; accepted 9 January 2003

Abstract The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) share about 90% identity at the genomic level but they have non-overlapping host range and show a high degree of host specificity. We have demonstrated here that AcMNPV undergoes DNA replication and early gene expression in Bombyx -derived BmN cells but fails to show very late gene expression or produce budded virion (BV) particles. Coinfection with BmNPV supported BV production from AcMNPV in BmN cells at low levels but not very late gene expression or polyhedral inclusion body formation. BV production and very late gene expression from BmNPV, on the contrary, were adversely affected in coinfections. In Spodoptera frugiperda -derived Sf21 cell lines, BmNPV DNA replication, BV production, and very late gene expression took place only when coinfected with AcMNPV. BmNPV exerted a less profound effect on AcMNPV multiplication and very late gene expression in permissive host cell lines. AcMNPV shuts down cellular and viral protein synthesis completely when infected alone or coinfected with BmNPV in BmN cells, whereas BmNPV infection did not affect cellular and viral protein synthesis in Sf21 cells. Overall, AcMNPV showed a more dominant effect by complementing the multiplication of BmNPV in nonpermissive host cells while inhibiting it in BmN cells. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Baculovirus; AcMNPV; BmNPV; Host range; Virus infection process

1. Introduction The Baculoviridae family of viruses are infectious to Arthropods, particularly insects of the order Lepidoptera, Diptera, and Hymenoptera. Baculoviruses are classified into three major subgroups: nucleopolyhedrovirus (NPV), granulovirus (GV), and non-occluded baculoviruses, and are characterized by the presence of large, circular, supercoiled, double-stranded DNA genomes packaged into rod-shaped virions. In occluded baculoviruses two types of particles are produced during an infection cycle, the budded virions (BVs) for efficient spread between the cells within an infected host, and occluded virions (OVs) for spread from insect to insect in a population (Keddie et al., 1989). Over the past two decades, baculoviruses have become popular as efficient

* Corresponding author. Tel.:
/91-80-360-0090; fax:
/91-80-3602697. E-mail address: [email protected] (K.P. Gopinathan).

vector for the high-level expression of foreign genes (Miller, 1988; Maeda, 1989, 1995; Possee, 1997) and safe alternatives to traditional chemical insecticides because of their ability to control specific insect pests in agriculture and forestry (Miller, 1995; Bonning and Hammock, 1996; Thiem, 1997). Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV), the prototype baculovirus, has the ability to infect 39 species of Lepidopteran larvae belonging to 13 families (Gershburg et al., 1997). The availability of appropriate tissue culture systems and detailed molecular studies has facilitated the isolation of genetically engineered AcMNPVs with novel properties. Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen of the mulberry silkworm B. mori , and causes serious economic damage to silk production. Sequence analysis of BmNPV and AcMNPV genomes revealed that they have over 90% identity (Gomi et al., 1999). Despite the high degree of homology between these viruses, their host ranges are

0168-1702/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1702(03)00046-7

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Md.M. Rahman, K.P. Gopinathan / Virus Research 93 (2003) 13 /23

non-overlapping. BmNPV replicates in the B. mori derived BmN cell line but not in Spodoptera frugiperda derived Sf21 or Sf9 cell lines which are permissive for AcMNPV. The converse of the host cell specificity also holds true. Elucidation of host cell specificity of baculoviruses is important for safe application of viruses in the field. The host range of any virus is determined by its ability to enter the cells and tissues of a susceptible host, and to replicate and release new infectious virus particles. The major steps in the baculovirus life cycle are the entry of virus into the cell, the viral early, late, and very late gene expression, DNA replication, budded virus assembly and release, and polyhedral inclusion body (PIB) formation. The budded form of the virus is known to enter the host cell by adsorptive endocytosis which involves the viral surface glycoprotein GP64 or LD130 homolog (Pearson and Rohrmann, 2002). Following entry into the cell, baculoviral nucleocapsid moves to the nucleus where the viral DNA replication depends on early gene expression, which is mediated by the cellular transcription machinery (Fuchs et al., 1983; Huh and Weaver, 1990). The early gene products include enzymes involved in viral DNA replication as well as transcriptional activators that modulate early/late gene expression. DNA replication is mandatory for late and very late gene expression (Thiem and Miller, 1989). Origins of DNA replication differ among baculoviruses (Kool et al., 1995). Although essential replication genes are conserved among baculoviruses, the genes contributing to optimal DNA replication from virus-specific DNA replication origins differ in different cell lines (Lu and Miller, 1995). Activation of late and very late gene transcription is also dependent on the induction of a new RNA polymerase activity that is insensitive to aamanitin (Huh and Weaver, 1990). Late gene products provide the structural components of BV and activate very late gene expression (Wilson et al., 1987; Thiem and Miller, 1989). Very late gene products are needed for the production of OV (Kuzio et al., 1989). Although several mechanisms operate in conferring host cell specificity, the nature of the block(s) to productive baculovirus infection in refractive insect cells is not fully understood. The involvement of DNA helicase in extending the host range of AcMNPV to permit its multiplication in BmN cells has been demonstrated (Maeda et al., 1993; Croizier et al., 1994). However, the converse situation, whether BmNPV multiplies in Sf cells if the corresponding helicases are swapped, is not known. Similarly, AcMNPV replication in LD652Y cells takes place only in presence of the product of hrf -1 (not related to DNA helicase) encoded by Lymantria dispar MNPV (Thiem et al., 1996). The synergistic or antagonistic effects of each of the viruses on the other in their respective permissive or nonper-

missive host cells have not been investigated. Here we report this latter aspect using BmNPV and AcMNPV as the model.

2. Materials and methods 2.1. Cells and viruses B. mori -derived BmN cells and S. frugiperda -derived Sf21 cells were maintained at 27 8C in TC-100 insect medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). A local isolate of BmNPV, BmNPV-BGL (Palhan and Gopinathan, 1996), and AcMNPV were routinely propagated in BmN and Sf21 cells, respectively. Recombinant BmNPV and AcMNPV (vBmluc and vAcluc ), harboring the reporter gene luciferase (luc ) in place of the polyhedrin (polh ) (Sriram et al., 1997), were used for very late gene expression monitoring purposes. Virus stocks were maintained and titered according to the standard protocols (O’Reilly et al., 1992). 2.2. Analysis of DNA synthesis by dot blot hybridization BmN and Sf21 cells (2.0 /104) were infected with virus at the appropriate multiplicity of infection (moi) in 96-well tissue culture plates, and DNA was isolated from cells in each of the wells. At the designated hours post infection (hpi), the cells were collected and stored at /80 8C. The frozen cells were lysed using 200 ml of 0.5 N NaOH, mixed with 1/10 volume of 3 M ammonium acetate, and blotted onto a nylon membrane (Hybond N
/, Amersham Pharmacia) using a 96-well dot blot manifold. The DNA was UV-crosslinked and hybridized to radiolabelled luc probe (labelled by random priming in presence of 32P-a-dATP) overnight at 42 8C in the presence of 50% formamide. After hybridization, the blots were washed at a final stringency of 0.1 / SSC (1 / SSC corresponds to 150 mM NaCl and 15 mM sodium citrate) and 0.1% sodium dodecyl sulfate (SDS) at 65 8C for 30 min, and the signals were visualized by autoradiography or phosphorimaging. 2.3. Assay of luciferase activity BmN and Sf21 cells (2.5 /105) were infected with virus at the moi indicated. The infected cells were harvested at 72 hpi, washed with phosphate buffered saline (PBS; 0.1 M sodium phosphate and 0.15 M NaCl, pH 7.4), and lysed using 1% Triton X-100. Luciferase activity in the clarified cell lysates (10,000 /g for 10 min) was quantified in a luminometer as relative luminescence units (RLU), as described previously (Sriram et al., 1997).

Md.M. Rahman, K.P. Gopinathan / Virus Research 93 (2003) 13 /23

2.4. Analysis of protein synthesis BmN or Sf21 cells (2.5 /105) in multi-well plates were infected with single or combination of viruses (moi: 10) and pulse-labelled for 1 h (O’Reilly et al., 1992) with 5 mCi of 35S-L-Met per 100 ml of Met-free culture medium at 0, 6, 12, 24, 36, 48, and 72 hpi. The cell pellets were washed free of medium, suspended in 20 ml of 1 / SDS gel loading buffer (Sambrook et al., 1989), and subjected to electrophoresis on 0.1% SDS-10% acrylamide gels (SDS-PAGE). Following electrophoresis, the gels were treated with sodium salicylate as enhancer for fluorography, dried under vacuum, and autoradiographed or alternately monitored by phosphorimaging. 2.5. Envelop fusion assay (Blissard and Wenz, 1992) Successful infection of insect cells with baculoviruses leads to cell fusion on exposure of the cells to low pH. BmN or Sf21 cells were seeded at a density of 1 /106 cells per 35 mm dish and infected with the viruses (AcMNPV, BmNPV, or both). At 48 hpi the medium was removed and the cells were treated for 5 min with TC-100 medium adjusted to pH 5.0. The cells were washed with fresh medium, incubated for 4 h at 27 8C, and observed microscopically for cell fusion.

3. Results 3.1. Virus production in permissive and nonpermissive cell lines In order to readily differentiate between the BmNPV and AcMNPV progeny emerging from the infection process, a wild-type virus (with intact polh and consequently resulting in the formation of PIBs) and a recombinant virus (where polh was replaced by luc ) were used in all the mixed infection studies. BmN cells when infected with BmNPV (wild-type or recombinant vBmluc viruses) exhibited rounding, swelling, and nuclear expansion by 60/72 hpi (Fig. 1, panel A, compare (b) and (d) with control cells shown in (a)). As expected, no PIB formation was seen in vBmluc infection (d). About 80 /90% of cells showed PIB formation on infection with BmNPV (b), which was reduced significantly when coinfected with AcMNPV ((e); compare with (b), see also Table 1). Infection of BmN cells with AcMNPV alone did not show any cytopathic effects or PIB formation (c). Although the cell morphology appeared similar to that of uninfected cells, cell multiplication was arrested. Coinfection of BmN cells with AcMNPV and BmNPV did not support AcMNPV multiplication as evidenced from the absence of PIB formation from wild-type AcMNPV (f). These results indicated that BmNPV did not complement

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AcMNPV multiplication in BmN cells, but the coinfecting AcMNPV in fact caused a reduction in PIB formation from BmNPV (Table 1). In parallel experiments in which Sf21 cells were used as host for infection with BmNPV and AcMNPV, either alone or together, the results were significantly different. Sf21 cells are permissive for AcMNPV and exhibited rounding, swelling, and nuclear expansion as well as PIB formation by 60 /72 hpi when infected with AcMNPV (Fig. 1, panel B, (b) and (d)). Formation of PIB was reduced to about 60% of cell population when coinfected with BmNPV ((e); see also Table 1). Sf21 cells did not show cytopathic effects when infected with BmNPV alone ((c); compare to (b)). Morphology of the cells was similar to that of uninfected Sf21 cells (a) and growth was not arrested. Coinfection of Sf21 cells with BmNPV and AcMNPV resulted in BmNPV multiplication as evidenced by PIB formation (f). These observations were quantified and are summarized in Table 1. To examine the effect of coinfection on BV production, the culture supernatants from BmN and Sf21 cells infected with the viruses were titered (Table 2). When BmN cells were coinfected with BmNPV and AcMNPV, the titer of the former was reduced by 20- to 40-fold (compare rows 1 and 5 in the third column). The very low basal levels of AcMNPV or BmNPV seen in the nonpermissive host cell lines (1 /104 /4/104 pfu/ml) were the background due to nonspecific sticking of virus to the cells. However, AcMNPV titers increased by 40to 50-fold over this background when coinfected with BmNPV (compare rows 3 and 5 in the last column). BmNPV thus complemented AcMNPV multiplication in BmN cells. Similarly, the BmNPV titers on propagation in Sf21 cells increased 1000- to 4000-fold over the background levels when coinfected with AcMNPV (Table 2; third column, compare rows 2 and 6). AcMNPV thus showed a more profound effect on BmNPV production. Coinfection of the two viruses in Sf21 cells reduced the titer of AcMNPV but only by 2- to 10-fold (compare rows 4 and 6). The inhibitory effect of BmNPV on AcMNPV budded virus production was much less pronounced, consistent with PIB production. 3.2. Expression of viral early genes Expression of one of the viral early genes, gp 64, was monitored by a functional assay. Baculovirus surface glycoprotein GP64, synthesized in the infected cells from early to late stages, is responsible for low pH-mediated cell fusion. BmN and Sf21 cells were infected with the viruses alone or in combination, and cell fusion following exposure of the cells to pH 5.0 was monitored after 24 hpi. BmN cells when infected with AcMNPV showed fusion to the same extent as seen with the permissive BmNPV infection (Fig. 2b and c, respectively; compare

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Md.M. Rahman, K.P. Gopinathan / Virus Research 93 (2003) 13 /23

Fig. 1. Cytopathic effects in infected cells. The morphology of BmN (panel A) and Sf21 (panel B) cells at 72 hpi, following infection or coinfection (moi: 10) with BmNPV and AcMNPV. In panel A, BmN cells uninfected (a) or infected with BmNPV (b), AcMNPV (c), vBmluc (d), both BmNPV and vAcluc (e), and both AcMNPV and vBmluc (f) are presented. In panel B, Sf21 cells uninfected (a) or infected with AcMNPV (b), BmNPV (c), vAcluc (d), both AcMNPV and vBmluc (e), and both BmNPV and vAcluc (f) are presented. Arrows indicate polyhedra-containing cells. Magnification: /200.

to Fig. 2a, uninfected cells). BmN cell fusion mediated by AcMNPV infection was also seen even in presence of the DNA replication inhibitor aphidicolin (data not shown), as anticipated for the early gene product

(GP64)-mediated fusion property. Sf21 cells, on the other hand, did not show fusion when infected with BmNPV (Fig. 2f; compare with Fig. 2e, showing Sf21 cells infected with AcMNPV) and appeared like the

Md.M. Rahman, K.P. Gopinathan / Virus Research 93 (2003) 13 /23

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Table 1 Production of PIBs from BmNPV and AcMNPV Host cell line

BmN Sf21

Number of host cells (per 1000) showing PIB formation following infection BmNPV alone

AcMNPV alone

BmNPV
/vAcluc

AcMNPV
/vBmluc

856 0

0 802

78¡/ 134/

0 507¡/

BmN or Sf21 cells were infected with BmNPV or AcMNPV (wild-type, or recombinant viruses vBmluc or vAcluc to readily distinguish the progeny) alone or in combination, and the PIB formation was monitored microscopically. PIB formation is due to the wild-type virus population in each sample. The downward and upward arrows indicate a decrease or increase in PIB in coinfections.

uninfected Sf21 cells (Fig. 2d). The possibility that BmGP64 has been synthesized but is incapable of causing cell fusion in Sf21 cells was ruled out by monitoring the synthesis of GP64 in these cells using specific antibodies raised against the protein. We could not detect the protein in BmNPV-infected Sf21 cells while GP64 synthesis was clearly detected in BmNPVinfected BmN cells or AcMNPV-infected Sf21 cells. 3.3. Viral DNA replication Viral DNA replication in permissive and nonpermissive host cells following single or combined infection with BmNPV and AcMNPV was monitored. Once again, by using a combination of wild-type and recombinant (vBmluc or vAcluc ) viruses, the replication of the two viruses could be differentially monitored using luc as a probe for hybridization (Fig. 3a and b). The radiolabelled luc probe will pick up signals only due to the recombinant virus (vBmluc or vAcluc ) DNA and not the wild-type viral DNA. The wild-type BmNPV DNA or host cell DNA was not detected (rows 2 and 1). The sensitivity of the probe was good enough to detect 1 /10 ng DNA (see lower most rows in Fig. 3a and b). BmNPV DNA contents increased by four- and twofolds at the two different moi analyzed at early times (12 and 24 hpi, respectively) but the levels were equal at later times, as quantified from the phosphorimager. BmN cells, though nonpermissive for AcMNPV, allowed its

replication from early to late times at both mois tested (Fig. 3a, rows 3 and 4). However, at high moi of coinfecting BmNPV, nearly 35% inhibition of AcMNPV replication was evident (row 5). BmNPV DNA replication in BmN cells was also confirmed in parallel experiments (rows 6 and 7) which showed no significant effect with coinfecting AcMNPV at the two different mois (rows 8 and 9). BmNPV did not replicate in its nonpermissive Sf21 cell line (Fig. 3b, row 3). However, when coinfected with AcMNPV, BmNPV showed replication in Sf21 cells at both mois tested (rows 4 and 5). As expected, AcMNPV replicated in its cognate host cells (rows 6 and 7), and coinfection with BmNPV at two different mois had no significant effect on AcMNPV replication (rows 8 and 9), akin to the results seen on coinfection in BmN cells described above. 3.4. Expression of viral very late genes Expression of the viral very late genes was monitored as luciferase activity from the polh promoter. BmN and Sf21 cells were infected either singly or in combination with the viruses at moi of 1 or 5 and the luciferase activity was determined at 72 hpi (Fig. 4). Very high levels of luciferase activity were seen even at moi of 1 (5 /106 /9 /106 RLU) in the permissive host cells with the corresponding viruses (Fig. 4a and b). Coinfection of vBmluc with AcMNPV in BmN cells resulted in 20- to

Table 2 Production of budded virus from BmNPV and AcMNPV Virus inoculum (moi: 5)

Propagating cells

BV titer (pfu/ml) from propagating host BmNPV

AcMNPV

BmNPV alone

BmN Sf21

6.8/107 1.4/104

/ /

AcMNPV alone

BmN Sf21

/ /

4.2 /104 1.4 /108

BmNPV
/AcMNPV coinfection

BmN Sf21

3.6/106 1.1/107

1.5 /106 5.6 /107

The virus infections, alone or in combination, were carried out in BmN and Sf21 cells, respectively. The emerging BV titers were determined at 72 hpi as TCID50 in the appropriate permissive cell lines. The experiments were repeated three times independently and the values shown here are from a typical experiment. The extent of variations is mentioned in the text. pfu, plaque forming units.

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Fig. 2. Early gene (gp 64) expression in infected cells monitored by fusion assay. BmN (a /c) and Sf21 (d /f) cells were infected with BmNPV (c, f) or AcMNPV (b, e) at moi 10. After 24 hpi, the cells were exposed to TC-100 medium at pH 5.0 for 5 min, washed and incubated with fresh TC-100 complete medium, pH 6.2 for 4 h, and observed microscopically. Arrows indicate the aggregation of cells (nuclei within syncytia). Uninfected cells (a, d) subjected to the same treatment. Magnification: /200.

100-fold reduction in luciferase levels from BmNPV (Fig. 4a). Infection of BmN cells with vAcluc alone or together with BmNPV at two different mois did not show any luciferase activity (Fig. 4a). Similarly, coinfection of Sf21 cells with vAcluc and BmNPV also resulted in the reduction of luciferase levels but only by 50 /75% at increasing moi of BmNPV (Fig. 4b). Although single infection of vBmluc in Sf21 cells did not show any luciferase activity, coinfection with AcMNPV resulted in fair levels of expression from the BmNPV pol h promoter (Fig. 4b). However, increase in moi of the coinfecting AcMNPV to 5 caused some decrease in the expression. Thus, it is apparent that AcMNPV exerted a more dominant effect on the very late gene expression from BmNPV, stimulating it in the nonpermissive host cell line and inhibiting it in the permissive cell line. Conversely, BmNPV showed less pronounced effect on late gene expression from AcMNPV in Sf21 cells and in fact, expression from BmNPV polh promoter was stimulated by AcMNPV in Sf21 cells. 3.5. Protein synthesis The synthesis of proteins in BmN (Fig. 5a/c) and Sf21 cells (Fig. 5d/f) infected with the viruses (moi: 10) was analyzed. In BmNPV-infected BmN cells, the expression of host proteins was inhibited by 24 hpi. Between 48 and 72 hpi, nearly all host- and even virus-

specific protein syntheses were minimal except for the highly expressed 30-kDa band (marked by arrow head) corresponding to polyhedrin (Fig. 5a). On coinfection with AcMNPV, the inhibition of protein synthesis (both host- and virus-specific proteins) was more pronounced from 24 hpi (Fig. 5b). In fact, by 12 hpi itself the protein synthesis had significantly dropped. Even the 30-kDa band corresponding to polyhedrin was not detectable in these coinfections. Infection with AcMNPV alone was sufficient to cause an inhibition in protein synthesis in BmN cells almost as efficiently as in coinfection (Fig. 5c). Besides, AcMNPV infection blocked protein synthesis even from the infecting BmNPV. The inhibition of protein synthesis in Sf21 cells infected with AcMNPV alone or together with BmNPV was similar. The intense 30-kDa band corresponding to polyhedrin was present from 36 to 72 hpi (Fig. 5d and e). However, Sf21 cells infected with BmNPV alone did not show any changes in the protein synthesis and the patterns were comparable to uninfected cells (Fig. 5f). BmNPV, therefore, did not noticeably affect protein synthesis in the nonpermissive Sf21 cell line or from the coinfecting AcMNPV.

4. Discussion We have analyzed different events such as DNA replication, early gene expression, very late gene expres-

Md.M. Rahman, K.P. Gopinathan / Virus Research 93 (2003) 13 /23

Fig. 3. Viral DNA replication. Total DNA was isolated from BmN (A) and Sf21 (B) cells infected singly or in combination with AcMNPV, BmNPV, vAcluc , and vBmluc at different hpi as marked. DNA was fixed on to a nylon membrane and hybridized with 32P-adATP-labelled luc probe. Numbers in parentheses show the moi of the infecting virus. Known amounts of luc DNA was used for monitoring the sensitivity of the probe (bottom rows, as indicated). The rows are marked on the left side of the panel.

sion, budded virus production, and cellular protein synthesis in cell lines following infection by BmNPV and AcMNPV alone or in combination. The overall effects of the two viruses coinfecting in the respective permissive or nonpermissive cell lines are summarized in Table 3. In BmN cells, AcMNPV undergoes DNA replication and early gene expression, but the production of infectious BV or very late gene expression was not detected. When coinfected with BmNPV, however, there were low levels of BV production from AcMNPV. Very late gene expression (as monitored by expression of luc from the pol h promoter or the PIB formation) from AcMNPV was not supported by BmNPV. In contrast, BV production and late gene expression from BmNPV

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in its own permissive host cell BmN were inhibited by AcMNPV. In Sf21 cells, on the other hand, AcMNPV supported DNA replication, infectious BV production, and late gene expression from BmNPV. The coinfecting BmNPV also inhibited very late gene expression or BV production from AcMNPV in Sf21 cells to some extent. Viral DNA replication is a prerequisite for transcription of baculovirus late genes which encode the structural proteins of the virus (Blissard, 1996). AcMNPV DNA replicated in nonpermissive BmN cells although there was no production of infectious BV, suggesting that the viral early gene products executing DNA replication were synthesized by the host transcription machinery that was common for both viruses. Expression of early gene products was also evident as seen from the low pH-mediated cell fusion in AcMNPV-infected BmN cells. The early protein GP64, synthesized from as early as 6 hpi by host RNA polymerase, is responsible for this fusion (Blissard and Wenz, 1992; Jarvis and Garcia, 1994; Rahman and Gopinathan, unpublished results). Conversely, even the early steps of BmNPV infection were not supported in Sf21 cells. For instance, in Sf21 cells infected with BmNPV, GP64 protein was not synthesized although the promoters and transcription profiles of BmNPV gp 64 were nearly identical to the AcMNPV counterpart (Rahman and Gopinathan, unpublished results). This observation was also consistent with the absence of DNA replication and BV production from BmNPV in these cells. Although coinfection of AcMNPV reduced BV titer from BmNPV in BmN cells, the viral DNA replication itself was not significantly affected. Moreover, BmNPV DNA replicated to a similar extent in Sf21 cells when coinfected with AcMNPV. There was also BV production from BmNPV in Sf21 cells when coinfected with AcMNPV. Even a low moi of 0.2 of AcMNPV was sufficient to support BmNPV replication (data not presented). These results ruled out the possibility that BmNPV did not enter the Sf21 cells and suggested that BmNPV DNA replication required some helper functions from AcMNPV. AcMNPV has been known to support DNA replication of Spodoptera exigua MNPV in nonpermissive Sf21 cells when coinfected (Yanase et al., 1998). Earlier reports (Kondo and Maeda, 1991; Maeda et al., 1993) have shown that recombinant AcMNPV arising from mixed infections had the capacity to replicate in BmN cells. To the extent, of progeny viruses that we have analyzed for the presence of recombinants in the present study, we did not see any. However, the recombination frequency being generally low (0.1 /1.0% in these viruses) we cannot totally rule out the possible presence of recombinants. In both AcMNPV and BmNPV, p143, homologous to DNA helicases, is essential for DNA replication. A recombinant AcMNPV bearing a small region of the p 143 from

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Fig. 4. Very late gene expression in infected cells. BmN (a) and Sf21 (b) cells were infected with the viruses as indicated. After 72 hpi, the luciferase activity in the cell lysates was assayed. The luciferase assay system contained 10% of the clarified cell lysate and the assay buffer (30 mM tricin, 3 mM ATP, 15 mM MgSO4, and 10 mM DTT, pH 7.8) containing 10 mM luciferin. The light emission was monitored in a luminometer and expressed as RLU. Numbers shown in parentheses are moi of viruses used.

BmNPV within the AcMNPV counterpart replicated in both Sf9 and BmN cell lines (Maeda et al., 1993; Croizier et al., 1994). The conferment of the extended host range to AcMNPV (its acquired capacity to multiply within BmN cells) was attributed to the changes in serine and phenylalanine residues at positions 564 and 577, respectively (S564N and F577L; Argaud et al., 1998), but how these changes in p143 alter AcMNPV host range is unknown. The fact that the replacement or modification of the AcMNPV helicase with the BmNPV counterpart permitted replication of AcMNPV in BmN cells implicated the involvement of some of these domains in its interaction with host-specific factors. The converse situation whether BmNPV replicates in

Sf21 cells if the helicase domains are swapped between the two viruses is not established. Another major component of DNA replication, the proliferating cell nuclear antigen (PCNA) present on the AcMNPV genome, is absent from BmNPV. Indeed, BmNPV makes use of the host counterpart of PCNA for its replication with no apparent deficiencies in the replication process (Udupa and Gopinathan, unpublished results). The other genes that influence baculovirus host range include cell line-specific late transcription factors and apoptotic suppressors. The inhibition of AcMNPV infection in LD652Y cells could be overcome by hrf -1 gene product (unrelated to helicase) from L. dispar MNPV (Thiem et al., 1996; Du and Thiem, 1997).

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Fig. 5. Protein synthesis in infected cells. BmN (a /c) and Sf21 (d /f) cells were infected with BmNPV (a, f), AcMNPV (c, d), or coinfected with BmNPV and AcMNPV (b, e) at moi 10. Protein synthesis was monitored by pulse labelling with 35S-Met at different hpi as indicated. The arrowheads indicate the position of polyhedrin. Protein size markers are indicated on the left.

Table 3 Summary of events in permissive and nonpermissive host cell lines Propagating host

BmNPV

AcMNPV

BmNPV
/AcMNPV coinfection BmNPV

AcMNPV

Early gene expressiona

BmN Sf21


/ /


/
/

ND ND

ND ND

Viral DNA replicationb

BmN Sf21


/ /


/
/


/
/


/
/

BV productionc

BmN Sf21


/
/
/ /

/
/
/
/


/
/
/
/


/
/
/
/

Very late gene expressiond

BmN Sf21


/
/
/ /

/
/
/
/


/
/
/

/
/
/

ND, not determined. a In early gene expression,
/ and / represent presence or absence of cell fusion, respectively. b In viral DNA replication,
/ and / represent presence or absence of detectable DNA, respectively. c In BV production,
/
/
/ indicates
/107 PFU/ml;
/, 105 /106 PFU/ml; and /, no virus production. d In very late gene expression measured as luciferase activity,
/
/
/ indicates
/5 /106 RLU;
/
/, 1/106 /5 /106 RLU;
/, B/1 /105 RLU; /, no luciferase activity.

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Very late gene expressions (PIB production as well as luciferase expression from polh promoter) from BmNPV in nonpermissive Sf21 cells were also seen when coinfected with AcMNPV. AcMNPV provided the helper function presumably by sharing some viral factors because late gene expression from AcMNPV itself was inhibited by about 50%. BmNPV, on the other hand, did not support very late gene expression from AcMNPV in BmN cells although AcMNPV DNA had replicated and low levels of BV were produced. In fact, coinfection drastically reduced late gene expression from BmNPV in BmN cells. Therefore, the interaction of the virally encoded RNA polymerase subunits involved in late gene expression (reviewed by Acharya et al., 2002) with host cell-specific factors appears to be necessary for bringing out very late gene expression from the virus. Variabilities in the host range are also known in different isolates of AcMNPV. For instance, in presence of AcMNPV isolate L1, BmNPV replicated to give titers of 106 pfu/ml in BmN cells in agreement with our results reported here, but AcMNPV OT2 inhibited BmNPV replication completely (Kamita and Maeda, 1993). AcMNPV infection in BmN cells led to the shut down of host- as well as viral protein syntheses after 12 hpi. These results were in general agreement with the earlier studies showing that AcMNPV infection in nonpermissive BmN or LD652Y cells resulted in viral- and host protein syntheses shutdown (Kamita and Maeda, 1993; Guzo et al., 1992). In the two different cell lines, inhibition of viral replication and late gene expression occurred by direct or indirect inhibition of translational machinery. BmNPV did not arrest protein synthesis in nonpermissive Sf21 cell line, and this lack of inhibition of protein synthesis could be responsible for the less pronounced effect on the coinfecting AcMNPV. In fact, both Sf21 and BmN cells supported the full replication cycle of the noncognate viruses resulting in BV production but the late and very late events were most significantly affected. p35, an antiapoptotic gene encoding an inhibitor of cysteine proteases of the CED-3/ICE family caspases (Bump et al., 1995) in AcMNPV, is required for efficient infection of S. frugiperda larvae and Sf21 cells (Clem et al., 1991). However, p 35 deletion did not impair replication of AcMNPV in Trichoplusia ni larvae or in TN368 cells derived from it (Clem and Miller, 1993). Similarly, the presence of p 35 was not sufficient to prevent apoptosis in AcMNPV-infected Spodoptera littoralis -derived SL2 cell lines (Chejanovsky and Gershburg, 1995) or Choristoneura fumiferana -derived FPMICF-203 cell lines (Palli et al., 1996). The absence of p 35 function was compensated by apoptosis inhibitor genes (iap genes) from Orgyia pseudotsugata NPV (Birnbaum et al., 1994). The apoptotic properties of the BmNPV p 35, despite 90% identity to its AcMNPV counterpart, were distinctly different (Morishima et al., 1998).

The host cell-specific factor gene, hcf -1, from AcMNPV was necessary for successful infection of TN368 cells and to some extent for the enhancement of the infectivity of virus in T. ni larvae, but it was not necessary for virus replication or infectivity in Sf cells or Spodoptera larvae (Lu and Miller, 1996). It has also been suggested that an immune response restrict systemic infection of AcMNPV in a nonpermissive host, Helicoverpa zea (Washburn et al., 1996). The initial infection in midgut tissues was similar to that in susceptible larvae but the infection was cleared over time from midgut tissues, whereas in immunologically compromised H. zea larvae, the virus spreaded systemically. Evidently, the productive infection in nonpermissive cell lines is abrogated in a number of different ways involving both host and viral components. The functional interaction of virally encoded gene products with specific host cell components to execute viral DNA replication and late gene expression, as well as the modifications of the host cell translational machinery are crucial for successful viral multiplication but no obvious common restriction point could be identified so far. Elucidation of this information is important for better assessment of risk associated with the utilization of recombinant baculoviruses as insecticides due to their potential spread into nontarget organisms. This is especially relevant in geographic regions where insects such as the silkworms are cultivated, and the use of baculoviruses as biocides is practiced. The real situation in the fields may be more complex than that seen in the laboratory conditions.

Acknowledgements We thank the Department of Biotechnology, Government of India, and the Indo-French Centre for Promotion of Advanced Scientific Research (CEFIPRA) for financial support. M.M.R. is a recipient of SAARC (South Asian Association for Regional Cooperation) Fellowship and K.P.G. is Emeritus scientist of the Council of Scientific and Industrial Research (CSIR).

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