Factors regulating baculovirus late and very late gene expression in transient-expression assays.

0022-538X/96/$04.0010

Copyrightq1996, American Society for Microbiology

Factors Regulating Baculovirus Late and Very Late Gene

Expression in Transient-Expression Assays

JASON W. TODD,1,2_{A. LORENA PASSARELLI,}1_{ALBERT LU,}1,2

ANDLOIS K. MILLER1,2*

Department of Genetics1_{and Department of Entomology,}2

The University of Georgia, Athens, Georgia 30602

Received 26 June 1995/Accepted 29 December 1995

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transac-tivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the

vp39 promoter. To further define the factors required for late and very late promoter expression, we first

determined that the eighteen lefs were also required for expression from two other major baculovirus promot-ers: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient-expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhe-drosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.

Baculoviruses have novel molecular strategies for regulating the three major phases of transcription from their large dou-ble-stranded DNA genomes. Early gene transcription appears to be mediated by host RNA polymerase II (19, 44) and is influenced strongly by IE-1, the product of the viral gene ie-1, and further stimulated by IE-2, the product of the viral gene

ie-2, also known as ie-n (2, 10, 15, 21, 31, 43, 46–48). Late gene

transcription is mediated by a novel,a-amanitin-resistant RNA polymerase activity which is induced during virus infection (12, 13, 18, 19) and is probably encoded, at least in part, by the viral genome (40). Very late gene expression, which is required for occluded virus formation, is also mediated by ana -amanitin-resistant RNA polymerase but additionally requires the func-tion of a novel gene, vlf-1, which is predicted to encode a polypeptide with sequence motifs characteristic of a family of integrase/resolvases (28).

The promoters of most late and very late genes have novel properties, including an absolute dependence on a TAAG se-quence located at the initiation point of transcription (29, 32). Very late promoters appear to differ from late promoters by having an additional promoter element, referred to as the very late burst sequence, embedded within the DNA region speci-fying the untranslated leader of the very late transcripts (29, 32, 56). Understanding the novel mechanisms by which late and very late gene transcription is regulated is therefore of basic interest from a transcriptional perspective. It is also of applied interest, since baculovirus gene expression vectors usually em-ploy one of the two major very late promoters, those regulating P10 gene (p10) and polyhedrin gene (polh) expression (33).

We have previously identified 18 genes of Autographa

cali-fornica nuclear polyhedrosis virus (AcMNPV) (24, 26, 30, 35–

40, 51) which are necessary and sufficient for efficient transient expression from the promoter of the major late capsid protein gene, vp39 (29, 49), and are also involved in expression from the very late polh promoter (32) in SF-21 cells, a cell line established from the fall armyworm, Spodoptera frugiperda (54). All 18 of these genes affect the steady-state level of reporter gene transcripts (27).

Nine of the 18 genes (ie-1, ie-2, lef-1, lef-2, lef-3, lef-7, p143,

dnapol, and p35) are necessary and sufficient for efficient

rep-lication of transfected plasmid DNAs containing a putative origin of virus replication (27). The function(s) of ie-1 and ie-2 in these replication assays probably entails their ability to transactivate expression from early promoters which control transcription of other early viral genes required in the assay. The involvement of p143 and dnapol, which encode polypep-tides with sequence similarities to DNA helicases and DNA polymerases, respectively, suggests that late transcription in these assays is coordinated with DNA replication as it appears to be during viral replication (44, 49). The gene product of lef-3 has been shown to bind single-stranded DNA (16). The roles of

lef-1, lef-2, and lef-7 are also likely to be related to virus

origin-specific DNA replication, although their roles in this regard have been established only through transient-expression or plasmid replication analyses (20, 27). The involvement of p35, a gene known to block apoptosis in both invertebrate and vertebrate cells (5–9, 17, 41, 45), suggests that induction of apoptosis occurs during these transient assays in SF-21 cells and that effective inhibition of apoptosis is necessary to ob-serve both efficient DNA replication and gene expression (20, 27, 51).

The products of the nine other genes required for efficient transient expression from the late vp39 promoter (i.e., lef-4,

lef-5, lef-6, lef-8, lef-9, lef-10, lef-11, 39K, and p47) affect the

steady-state levels of reporter gene transcripts and are thus

* Corresponding author. Mailing address: Department of Entomol-ogy, University of Georgia, 413 Biological Sciences Bldg., Athens, GA 30602-2603. Phone: (706) 542-2294. Fax: (706) 542-2279. Electronic mail address: [email protected].

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likely to be involved in some aspect of transcription or tran-script processing and stability (27). This view is supported by the analysis of temperature-sensitive p47 and lef-4 mutants, which show reduced levels of late transcripts at the nonper-missive temperature (3, 4, 34). The 39K gene encodes a phos-phorylated protein, pp31, associated with the virogenic stroma, a virus-induced nuclear structure which appears to be the site of nucleocapsid assembly (14). The functional contributions of the remaining late expression factor genes (lefs) are unknown, although two of them, lef-8 and lef-9, encode polypeptides with sequence motifs conserved in many RNA polymerases (27, 40). An additional gene, very late factor 1 (vlf-1), is required to achieve optimal levels of very late transcripts but not late tran-scripts during virus infection (28). This gene was identified through the characterization of a temperature-sensitive mu-tant, tsB837 (23), which is defective in occluded virus produc-tion but produces normal levels of late RNAs and proteins in a timely manner (23, 28). The predicted product of vlf-1, VLF-1, is anticipated to have a novel mechanism of regulating gene expression because it possesses amino acid sequence sim-ilarity to a family of bacterial and yeast integrase/resolvases, including lambda phage integrase. It is important to determine whether vlf-1 exerts a transactivating effect in these transient-expression assays, since these assays would provide a powerful means of exploring the nature of VLF-1 and could also be used to search for additional very late expression factors.

In this study, we determined whether the 18 lefs are required for expression from two other promoters representative of the late and very late classes of transcription and whether vlf-1 exerted a specific effect on the very late class of promoters in transient-expression assays. The late p6.9 promoter drives ex-pression of a basic 6.9-kDa protamine-like protein (P6.9) which is associated with viral DNA within the nucleocapsid, forming the nucleoprotein core of virus particles (53, 58). Very late p10 encodes an abundant protein of 10 kDa which forms large fibrillar structures in both the cytoplasm and nucleus of in-fected cells during the very late phase of infection (55, 57). We found that the same 18 genes involved in expression from the

vp39 and polh promoters are also required for efficient

expres-sion from the p6.9 and p10 promoters in our

transient-expres-sion assay. Moreover, we show that vlf-1 is required for optimal expression from the very late p10 and polh promoters but not for expression from the late promoters in this assay. We also surveyed the AcMNPV genome for the presence of additional

vlf genes, but none were detected, suggesting that vlf-1 is

pri-marily if not exclusively responsible for activating very late promoters. Because of the potential influence of post-transfec-tion harvest time on reporter gene expression, we also exam-ined this parameter and found that the influence of p35 varies both with regard to the time posttransfection and with the nature of the viral genes provided in the cotransfection mix-ture.

MATERIALS AND METHODS

Cells and virus.The cell line IPLB-SF-21 (SF-21) (54), derived from the fall armyworm, S. frugiperda, was used for transfections. SF-21 cells were grown at 278C in TC-100 medium (GIBCO BRL, Gaithersburg, Md.) containing 10% fetal bovine serum and 0.26% tryptose broth (33). AcMNPV L-1 (22) was used as the source of wild-type viral DNA used in transfections.

Transfections and transient-expression assays.All transfections were per-formed with calcium phosphate (33) to coprecipitate 2mg of reporter plasmid, approximately 0.5mg of any other DNA, and salmon sperm DNA to standardize the total concentration of DNA per reaction in each experiment (35).

Protein lysates were collected at 48 h after cotransfection for samples con-taining the late reporter plasmids pCAPCAT and p6.9hc or 72 h after cotrans-fection for samples containing the very late reporter plasmids phcwt and p10hc, unless otherwise noted. Chloramphenicol acetyltransferase (CAT) assays (12a, 42) were done with between 1 and 10ml of a 100-ml lysate unless otherwise specified.

Reporter plasmids.The previously described reporter plasmids pCAPCAT (50) and phcwt (42) contain the vp39 and polh promoters, respectively, control-ling the reporter gene cat.

Plasmids p6.9hc and p10hc contain the promoters of the late P6.9 gene, p6.9, and the very late P10 gene, p10, respectively, controlling the reporter gene cat. These plasmids were constructed by digesting phcwt with BglII and EcoRV to remove a 92-bp fragment containing the polh promoter and inserting the p6.9 or

p10 promoter in its place. The p6.9 and p10 promoters were obtained by PCR.

The p6.9 promoter contains the region from11 to2159, while the p10 promoter contains the region from11 to2108, relative to their respective ATGs. The PCR products were cut with BglII and EcoRV and gel purified prior to their insertion into phcwt. Thus, all late and very late reporter plasmids contain cat within the polh locus flanked by a portion of the hr5 sequence (42).

Other clones and plasmids.The plasmids and lambda clones used to supply the wild-type and mutant forms of specific genes or sets of genes are summarized in Table 1. The complete AcMNPV library of genomic clones included all the

TABLE 1. Genes and plasmids affecting late and very late gene expression

Gene Plasmid Mutated form Reference Functiona

Genomic clone(s)b

lef-1 pBCNE 35 DNA replication BC5

ie-1 pIE1/HC 37 trans activator IE15

ie-n pPstN 37 trans activator IE15

lef-2 p630 p630del 37 DNA replication IE15

lef-3 pSDEM2 24 DNA replication PstH5

p143 pR1D/SdelORF5 36 DNA replication (helicase motif) HC10

lef-4 86D5B 86D5BdAccI 36 lef HC10

lef-5 pORF6 pORF6del 36 lef HC10

lef-6 pAcIAP-Ps/NsiI pIAPPsdelHxdelSal 38 lef BC5, HL8, HL5

lef-7 pBSXBglII pLEF7fs 29, 51 DNA replication pXmaB

lef-8 pRI-M pRI-M/MluI 40 RNA polymerase motif PstH4, ETL7

lef-9 pPstH1SB2.35 pPstH1SB2.35fsHdIII 26 lef PstH4, PstH1, PstH5

lef-10 pPstH1Sal0.8 26 lef PstH4, PstH1, ETL7

dnapol pDNAP pDNApdRI 26 DNA polymerase PstH1, PstH5

lef-11 pH3R or pH3R/NH pH3R/ORF5fs 51 lef HL8, HL5, ETL7

p47 p47 p47/XbaI 51 lef HL5, ETL7

39k pNspAfl or pFspAfl pFspAfl/fs 51 lef HL8, HL5, ETL7

p35 pRS pRSdel 11 Blocks apoptosis HK5

vlf-1 pXA7 pXAdBSAc _{28 Very late expression factor PstH5, HC9}

a

Function is inferred from involvement in transient-expression assays and DNA replication assays, temperature-sensitive mutations, or sequence motifs.

b

Clone(s) of the overlapping library of the AcMNPV genome containing the gene.

c

Construction of this clone is described in Materials and Methods.

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genomic clones listed in this table. The ‘‘lef’’ library consisted of all the plasmids supplying a lef function (Table 1, second column) except pXA7, which supplies

vlf-1. Plasmid pH3R was used to supply lef-11 in the experiment shown in Fig. 3, while pH3R/N was used in the experiment shown in Fig. 5; pFspAf1 was used to supply 39K unless otherwise noted. Plasmid pDNApdRI, not previously de-scribed, contains a frameshift mutation in dnapol at the EcoRI site (41.24 map units [m.u.]) 21 codons from the N-terminal end of the open reading frame (ORF). It was constructed by partially digesting pDNAp with EcoRI, filling in the ends with Klenow, and religating; the EcoRI-U and EcoRI-V fragments upstream of dnapol were deleted during cloning. Plasmid pXAdBSA was con-structed by digesting pXA7 (28) at a unique BstEII site within vlf-1, filling in the ends with Klenow fragment, and religating. The presence of a frameshift muta-tion within vlf-1 was confirmed by nucleotide sequencing.

RESULTS

Effect of omitting each AcMNPV library clone on expression

from the p6.9 and p10 promoters.To obtain an overview of the

viral factors required to transactivate the late p6.9 promoter and the very late p10 promoter, we first examined the effect of removing each of the 12 genomic library clones collectively representing the entire AcMNPV genome. Both the p6.9hc and p10hc reporter plasmids responded similarly; removal of the BC5, PstH1, HC10, and IE15 clones had strong effects on the level of expression from both reporter plasmids (data not shown). This result was similar to that reported previously for the late pCAPCAT reporter plasmid and for phcwt, a very late reporter plasmid (35). To define the genes involved in

trans-activation, the genes present in each of these regions of the genome were defined as described below.

lef-1, lef-2, lef-3, lef-4, lef-5, lef-7, p143, ie-1, and ie-2 are

required for optimal transient expression from the late p6.9

and the very late p10 promoters.Omission of any one of the

library clones BC5, PstH5, and HC10 resulted in a major drop in activity of the p6.9 and p10 promoters (Fig. 1A and B, compare lane 1 with lanes 2, 4, and 6). Substitution of pBCNE (35) containing lef-1 for BC5 restored activity to a level similar to that of the whole library (Fig. 1A and B, compare lanes 1 and 3). Similarly, substitution of pSDEM2 (24) containing lef-3 for PstH5 restores activity (Fig. 1A and B, compare lanes 1 and 5). The library clone HC10 contains three lefs: lef-4, lef-5, and

p143. Omitting HC10 while adding plasmids supplying these

three lefs, 86D5B, pORF6, and pR1D/SdORF5, respectively, produced activity similar to that with the entire library (Fig. 1A and B, lanes 1 and 7). Omitting or disrupting any of these three

lefs resulted in only background levels of activity (Fig. 1A

and B, compare lanes 1 and 7 with lanes 6 [HC10 omitted], 8 [pR1D/SdORF5 omitted], 9 [pORF6del, contains a deletion in

lef-5], and 10 [86D5BdelAccI, contains a frameshift mutation

in lef-4]). These data indicated that lef-1, lef-3, lef-4, lef-5, and

p143 encode important factors for transient expression from

the p6.9 and p10 promoters.

Omission of IE15 from the AcMNPV library resulted in a dramatic decrease in the activity of both the p6.9 and p10 FIG. 1. Determining the contribution of genes located within clones BC5, PstH5, HC10, IE15, and pXmaB on expression from the p6.9 and p10 promoters. Reporter plasmids p6.9hc (A) and p10hc (B) were cotransfected with the entire AcMNPV clone library (lane 1) or the library lacking one of the clones, as indicated below each lane. Plasmids supplying wild-type or mutated lefs located within each of the omitted clones were added as indicated above each lane. CAT assays were performed on cell lysates; acetylated products (Ac Cm) and unacetylated substrate (Cm) are indicated on the right.

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promoters (Fig. 1A and B, lanes 1 and 11); a similar decrease was seen with the vp39 and polh promoters (37). Three lefs are known to be located in the library clone IE15: ie-1, ie-2, and

lef-2 (37). A fourth gene, pe38, is reported to have a

stimula-tory effect on expression from some viral promoters (25, 39). Substituting plasmids p630 containing lef-2, pIE1/HC contain-ing ie-1, pPstN containcontain-ing ie-2 (37), and pPE38 containcontain-ing pe38 (39) restored activity to a level slightly higher than that with the entire library (Fig. 1A and B, lanes 1 and 12). If a clone containing a lacZ insertion in pe38, pPE38Z, was used instead of pPE38, no difference was seen with the p6.9 promoter (Fig. 1A, lanes 12 and 13), but a slight decrease was observed with the p10 promoter (Fig. 1B, lanes 12 and 13). Supplying pIE1/ HC, pPstN, and a clone with a deletion in lef-2, p630del, re-sulted in a significant drop in activity (Fig. 1A and B, compare lanes 13 and 14). Omitting pPstN (used to supply ie-2) while adding pIE1/HC and p630 resulted in a more modest decrease in activity from the p6.9 and p10 reporter plasmids (Fig. 1A and B, lanes 13 and 15), indicating that ie-2 stimulated expres-sion from these promoters but was not strictly required. When only p630 and pPstN were substituted for IE15 (i.e., when ie-1 was not supplied), no CAT activity was detected for either the

p6.9 or p10 promoter (Fig. 1A and B, lane 16), indicating a very

strong requirement for ie-1. The effects of the genes within IE15 on expression from the p6.9 and p10 promoter reporter plasmids were thus very similar to the effects observed with the

vp39 and polh promoter reporter plasmids (37).

Deleting pXmaB, which contains lef-7, resulted in a subtle decrease in activity from the p6.9 and p10 promoter reporter plasmids (Fig. 1A and B, lane 17). The decrease in activity could be compensated for by supplying pBSXBglII containing

lef-7 but not by pLEF7fs, which contains a mutation in lef-7

(Fig. 1A and B, lanes 18 and 19). The removal of pXmaB in the context of the rest of the AcMNPV clone library had a similarly subtle effect on expression from the vp39 and polh reporter plasmids (30).

lef-6, lef-8, lef-11, p47, and 39k are required for expression

from the late p6.9 and the very late p10 promoters.Several lefs

are located in the region of the AcMNPV genome included in the library clones BC5, HL8, HL5, ETL7, and PstH4. One of these is lef-6, which is contained within three library clones, BC5, HL8, and HL5. To assess the requirement for lef-6, these clones were omitted, and plasmids supplying the additional lefs in the region, lef-1 (pBCNE), 39k (pFspAfl), lef-11 (pH3R/ NH), and p47 (p47), were added (Fig. 2A and B, lanes 2 to 4). (lef-1 is contained within BC5 only, while 39k, lef-11, and p47 were added to ensure adequate copy number because all but one library clone supplying them were omitted [Table 1].) Ad-dition of a plasmid containing lef-6, pAcIAP-Ps/NsiI, restored activity (Fig. 2A and B, lane 3); this plasmid contained a frameshift in the upstream Ac-iap to ensure that Ac-iap was not responsible for the stimulatory activity. When a plasmid carrying a deletion in lef-6 (pIAP-PsdelHxdelSal) was added, only background activity was observed (lane 4), confirming the involvement of lef-6 in expression from these promoters.

Three lefs are known to be supplied only by the library clones HL8, HL5, and/or ETL7: 39k, lef-11, and p47 (51). In lanes 5 to 10 of Fig. 2A and B, these three library clones were omitted and plasmids pAcIAP-Ps/NsiI (lef-6) and pRI-M (lef-8) were added to avoid loss of activity due to dosage effect.

Adding p47 (p47), pH3R (lef-11), and pFspAfl (39k with the

lef-11 ORF intact) or p47, pH3R, and pNspAfl (39k with a partial lef-11 ORF) resulted in a major increase in activity over background (Fig. 2, compare lane 5 with lanes 6 and 7). As

FIG. 3. Effects of lef-9, lef-10, and dnapol on expression from the p6.9 and

p10 promoters. Reporter plasmids p6.9hc (A) and p10hc (B) were cotransfected

with the entire AcMNPV clone library (lane 1) or the library lacking clones PstH4, PstH1, and PstH5, as indicated below lanes 2 through 6; the loss of lef-3 (normally supplied by PstH5) was compensated for by its addition, as indicated below the bracket. Plasmids supplying dnapol (pDNAp, wild type; pDNApdRI, frameshifted), lef-9 (pPstHISB2.35, wild type; pPstHISB2.35HindIII, frame-shifted), or lef-10 (pPstHISal0.8) were added in the combinations indicated above each lane. CAT assays were performed on cell lysates; acetylated products (Ac Cm) and unacetylated substrate (Cm) are indicated on the right. FIG. 2. Effects of genes located within clones BC5, HL8, HL5, ETL7, and PstH4 on expression from the p6.9 and p10 promoters. Reporter plasmids p6.9hc (A) and p10hc (B) were cotransfected with the entire AcMNPV clone library (lane 1) or the library lacking one of the AcMNPV library clones as indicated below each lane. Plasmids supplying lef functions located within each of the omitted clones were added as indicated under the brackets and above each lane. CAT assays were performed on cell lysates; acetylated products (Ac Cm) and unacetylated substrate (Cm) are indicated on the right.

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FIG. 4. Effect of the AcMNPV antiapoptosis gene p35 on expression from the p6.9, p10, vp39, and polh promoters. The reporter plasmids (p6.9hc, p10hc, pCAPCAT, or phcwt) used in each experiment are indicated above the lanes. (A) Effects of omitting p35 from the AcMNPV clone library (p35 supplied by HK5 in lanes 1 and 8 but omitted in lanes 2 to 4 and lanes 9 to 11) or the lef library (p35 supplied by pRS) (lanes 5 to 7 and lanes 12 to 14) as indicated below the lanes. Additional plasmids supplied in each experiment are indicated above the lanes; pRSdel contains a deletion within p35. The lysates assayed in panel A were prepared from cells harvested at 48 h post-cotransfection for p6.9hc and at 72 h post-cotransfection for p10hc. Relative CAT activity is relative to the level of activity produced with the entire AcMNPV clone library (lane 1, 100%). (B) Effects of the time at which cells were harvested (noted in parentheses above the lanes) on the relative

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observed for the vp39 and pol promoters (51), these plasmids are unable to restore the full activity to the p6.9 and p10 promoters which was lost by omitting HL8, HL5, and ETL7. It is not clear whether this is due to the fact that the major 4.2-kb transcript for p47 and 39k (4) is not supplied intact by the smaller plasmids provided or if an unknown optimizing factor exists in this region. Evidence demonstrating that p47, 39k, and

lef-11 are needed for optimal expression from the p6.9 and p10

promoters is shown in Fig. 2A and B, lane 8 (p47/XbaI con-taining a frameshift in the p47 ORF was substituted for p47), lane 9 (pFspAfl/fs containing a frameshift mutation in 39k was substituted for pFspAfl), and lane 10 (pH3R/ORF5fs contain-ing a mutation in lef-11 was substituted for pH3R).

lef-8 is supplied by the library clones PstH4 and ETL7.

Omis-sion of these two clones resulted in a major drop in activity of the p6.9 and p10 promoters (Fig. 2A and B, lanes 1 and 11). Plasmids p47 and pPstH1Sal0.8 (lef-10) were added in lanes 11 to 13 to avoid loss of activity due to dosage effect. Adding pRI-M restored activity (lane 12), but substituting a clone containing a frameshift mutation within lef-8, pRI-M/MluI, produced only background levels of activity (lane 13).

lef-9, lef-10, and dnapol are required for expression from the

late p6.9 and the very late p10 promoters.In lanes 2 to 6 of Fig.

3A and B, PstH4, PstH1, and PstH5 were omitted from the library, and plasmid pSDEM2 containing lef-3 (supplied by PstH5) was added. Adding plasmids supplying the three lefs known to be present in this region, pPstHISB2.35 (26) con-taining lef-9, pPstHISal0.8 (26) concon-taining lef-10, and pDNAp (26) containing dnapol (52), restored p6.9 and p10 promoter activity to library levels (Fig. 3A and B, lane 3). Adding pPstH1SB2.35 and pPstH1Sal0.8 and a clone with a deletion in

dnapol, pDNApdRI, produced activity that was substantially

lower than the level achieved by adding all three intact lefs but was somewhat higher than background (Fig. 3A and B, lane 4). This indicated that dnapol was needed for optimal p6.9 and p10 promoter activity but was not absolutely required. A strong requirement for both lef-9 and lef-10 was also observed; re-placement of pPstHI5B2.35 with pPstHISB2.35fsHdIII (con-tains a frameshift mutation in lef-9 [26]) (Fig. 3A and B, lane 5) and omission of pPstHISal0.8, containing lef-10 (lane 6), resulted in background levels of activity.

Involvement of p35 in expression from late and very late

promoters.Baculovirus genes with antiapoptosis activity (i.e.,

AcMNPV p35, Op-iap, and Cp-iap) are known to have a four-to fivefold stimulafour-tory effect on expression from the vp39 pro-moter and a 10-fold effect on the polh propro-moter of AcMNPV in the context of the lef library (51). Similar levels of stimula-tion by p35 were also observed for the p6.9 and p10 promoters (Fig. 4A, lanes 5 to 7 and lanes 12 to 14). The effect of p35 was less striking in the context of the AcMNPV genomic library; when the sole library clone providing p35, HK5, was omitted from the AcMNPV library, expression from late reporter plas-mids pCAPCAT and p6.9hc harvested 48 h post-cotransfection decreased approximately twofold or less (Fig. 4A and B, lanes 1 and 2). However, expression from all late or very late pro-moters decreased five- to sixfold upon omission of HK5 in cells harvested at 72 h post-cotransfection (Fig. 4B, lanes 5 and 6, lanes 9 and 10, and lanes 13 and 14; Fig. 4A, lanes 8 and 9). In

all cases involving the AcMNPV clone library, substituting pRS, which contains p35, for HK5 produced activity at least 70% as high as that with the entire library (Fig. 4A, lane 10; Fig. 4B, lanes 7, 11, and 15). This effect was not observed with pRSdel containing a deletion within p35 (Fig. 4A, lane 11; Fig. 4B, lanes 8, 12, and 16). Thus, p35 affects expression from the

p6.9 and p10 promoters in a similar fashion as the vp39 and polh promoters, and the relative level of the observed effect

depends to some extent on the time that the cells are harvested for assay and the nature of the other viral genes provided in the cotransfection.

Stimulation of very late but not late gene expression by vlf-1.

Although vlf-1 transactivates very late gene expression during virus infection (28), the effect of vlf-1 in transient-expression assays has not been reported previously. In order to determine if vlf-1 exerts an effect in this assay and if it acts specifically as a very late factor, we used the two late and two very late reporter plasmids: pCAPCAT, p6.9hc, phcwt, and p10hc.

Two overlapping clones of the AcMNPV library, PstH5 and HC9, contain vlf-1 (28). Omission of these two clones from the AcMNPV library resulted in a substantial decrease in CAT activity for all four reporter plasmids (Fig. 5A, B, C, and D, compare lanes 2 and 3). PstH5 also contains lef-3 (23), so a plasmid containing lef-3, pSDEM2, was added in lanes 4 to 6 of Fig. 5. For both late reporters, pSDEM2 supported activity at least as high as that with PstH5 and HC9 in the presence of the remaining library clones (Fig. 5A and B, compare lanes 2 and 4). When we also added pXA7, a plasmid containing vlf-1, we saw no significant increase in expression from either pCAP-CAT or p6.9hc. This was observed both for cells harvested at 48 h post-cotransfection (Fig. 5A and B, lanes 4 and 5) and for cells harvested at 72 h post-cotransfection (data not shown). For the two very late reporters, the level of activity supported by pSDEM2 was only about 40% of the level with the entire library (Fig. 5C and D, compare lanes 2 and 4). When we also added vlf-1 (by adding pXA7), expression increased approxi-mately seven- to eightfold from both phcwt and p10hc (Fig. 5C and D, lanes 4 and 5).

In order to determine if the effect of pXA7 on expression from very late promoters was indeed due to vlf-1, plasmid pXAdBSA, which contains a frameshift mutation in vlf-1, was substituted for pXA7. For the late promoters, the level of activity supported by pXAdBSA at 48 h post-cotransfection was similar to the level supported by pXA7 (Fig. 5A and B, lanes 5 and 6). This was also true for similar experiments in which the cells were harvested at 72 h post-cotransfection (data not shown). For phcwt and p10hc, the levels of activity ob-tained with pXAdBSA were close to the levels observed in the absence of pXA7 and five- to sevenfold lower than the levels with pXA7 (Fig. 5C and D, lanes 4 to 6). This evidence indi-cated that vlf-1 stimulated expression from both very late pro-moters, polh and p10, but did not stimulate expression from either late promoter, vp39 or p6.9, even when harvested at 72 h post-cotransfection, consistent with in vivo observations with a temperature-sensitive vlf-1 mutant.

Survey of the AcMNPV genome for additional vlfs in

tran-sient-expression assays.The original survey of the AcMNPV

genome for late and very late expression factor genes failed to

contribution of p35 to expression from different reporter plasmids (pCAPCAT, p6.9hc, and phcwt, as indicated above the lanes). The reporter plasmids were cotransfected with the entire AcMNPV clone library with or without HK5, as indicated below the lanes. pRS supplied an active form of p35, whereas pRSdel supplied a deleted form of p35; the presence of these plasmids is indicated above each lane. The numbers below each lane indicate the percent relative CAT activity, as determined by PhosphorImager analysis (model 425E; Molecular Dynamics, Sunnyvale, Calif.) for dilutions of cell extracts so that the CAT assays were in the linear range of the assay (30% or less conversion of substrate to acetylated product). The data are representative of two or more similar experiments. The acetylated products (Ac Cm) and unacetylated substrate (Cm) are indicated to the right.

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detect vlf-1, possibly because the effect of this factor is milder than the effect observed for the lefs. We therefore investigated whether there were any additional vlfs present in the AcMNPV genome by systematically omitting individual or overlapping sets of clones from the AcMNPV library, replacing them with

lefs that were present in those clones, and quantifying the effect

on expression from pCAPCAT, p6.9hc, phcwt, and p10hc (Ta-ble 2). Because of the complexity of this assay, involving the addition of numerous plasmids, differences of less than approx-imately twofold were not considered significant. In general, we

would expect the presence of a vlf to be detected as a selective decrease in the level of expression from the very late but not the late reporter plasmids, indicating that additional genes other than lefs were present in the genomic clone omitted.

With the exception of the PstH5/HC9 overlap region, which contains vlf-1, no significant differences in expression levels relative to expression from each promoter in the presence of the complete AcMNPV clone library were observed for any of the four reporter plasmids when individual or overlapping sets of clones were omitted from the library and replaced by the FIG. 5. Effects of vlf-1 on transient expression from late and very late promoters. Reporter plasmids pCAPCAT (A), p6.9hc (B), phcwt (C), and p10hc (D) were cotransfected with intact AcMNPV DNA (L-1, lanes 1), the entire AcMNPV clone library (lanes 2), or the AcMNPV clone library lacking PstH5 and HC9 (lanes 3 to 6). Plasmid pSDEM2 was supplied in lanes 4 to 6 to compensate for the loss of lef-3. The effects of adding a plasmid (pXA7) containing an active version of vlf-1 (lanes 5) or a plasmid containing an inactive version of vlf-1 (pXAdBSA, lanes 6) were determined by CAT assay of cell lysates harvested at 48 h post-cotransfection for the late reporter plasmids (pCAPCAT and p6.9hc) and at 72 h post-cotransfection for the very late reporter plasmids (phcwt and p10hc). Relative CAT activity was determined by PhosphorImager analysis for dilutions of cell extracts so that the CAT assays were in the linear range of the assay (30% or less conversion of substrate to acetylated product). The data are representative of two or more similar experiments. The acetylated products (Ac Cm) and unacetylated substrate (Cm) are indicated to the right.

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corresponding lefs in those regions. The general decline in ex-pression when IE15 was omitted is probably due to the lack of PE-38, which exerts an approximately twofold effect on expres-sion in this assay. In the case of the PstH5/HC9 overlap, a substantial difference was observed for both phcwt and p10hc in the presence and absence of pXA-7, which contains vlf-1, but virtually no difference was observed for expression from the late reporter plasmids pCAPCAT and p6.9hc. This con-firms that the very late promoters are responding to a very late expression factor gene in this assay. (Although the effect of

vlf-1 is routinely observed in assays of this nature, there is some

variability in the relative response of very late promoters to

vlf-1 from assay to assay. We believe that this is due to the

quality of the plasmid or lambda DNA preparations.) The absence of very late promoter-specific effects for other genomic regions suggests that there are no additional very late expression factor genes that have a significant effect on expres-sion from these reporter plasmids.

DISCUSSION

We have further characterized the AcMNPV genes involved in transient late and very late gene expression in SF-21 cells. We have shown that all 18 genes originally identified by tran-sient-expression analysis with vp39 and polh promoter-based reporter plasmids are also involved in expression from the late

p6.9 and very late p10 promoters in this assay. These genes are

thus likely to encode general factors which are necessary for optimal late and very late gene expression in this cell line. For the two late promoters, the levels of expression observed with the lef library approached the levels of expression observed when all the AcMNPV genes were supplied as genomic clones. This indicates that we have identified the major transactivating genes in the AcMNPV genome for these promoters and that the factors required for promoter transactivation are the same. Furthermore, the influence of each of the genes is similar for

these promoters, although some of the 18 genes have differing degrees of influence.

We have also shown that the presence of a very late factor gene, vlf-1, stimulates expression from the two very late pro-moters, polh and p10, but has little or no effect on the two late promoters, vp39 and p6.9. The requirement for vlf-1 in very late gene expression was established in vivo through the char-acterization of a temperature-sensitive mutant which failed to produce occlusion bodies (28). The ability of the transient assay to detect the effect of vlf-1 on very late gene expression is important because it provides a means of monitoring the activity of modified forms of vlf-1. Recently, an in vitro tran-scription assay which appears to respond differently to late and very late promoters was described (59); it has not been shown, however, that this system will respond to VLF-1 or exog-enously added gene products.

The fact that vlf-1 is able to increase transient expression from plasmids with very late promoters but not those with late promoters also provides insight into the mode of action of VLF-1. VLF-1 is predicted to contain sequence motifs charac-teristic of a family of integrase/resolvases (28). Because VLF-1 can stimulate expression from phcwt but not from p6.9hc, which differ only in the approximately 100 nucleotides consti-tuting the promoter region upstream of cat, it is likely that VLF-1 acts directly on the promoter or that only very late promoters are responsive to the influence(s) exerted on the reporter plasmid by VLF-1. VLF-1 therefore appears to be an excellent candidate for the factor which interacts with the burst element of very late promoters.

Although we have quantitatively surveyed the entire AcMNPV genome for the presence of additional vlfs, we have found no indication that additional vlfs are present. We have also exam-ined the ability of the three genes contaexam-ined within the re-porter plasmid (ORFs 7, 9, and 10) to transactivate late and very late reporter plasmids lacking these genes (35) (data not shown). These ORFs do not appear to be transactivating fac-tors. On the basis of these data, it seems unlikely that there are additional vlfs in the AcMNPV genome. However, the use of transient-expression assays may have limitations in this type of search.

We have also investigated the influence of transfection time on the contribution of the antiapoptosis gene p35 to gene expression in this assay. The relative influence that this gene exerts on the levels of expression from late reporter plasmids depends on the time that the cells are harvested following cotransfection; the later the cells are harvested, the greater the influence p35 has on expression. Thus, cells harvested at 48 h post-cotransfection show a less than twofold stimulation by p35 in the presence of the rest of the AcMNPV genome, whereas cells harvested at 72 h post-cotransfection show at least a four-to sixfold effect. Although we have not demonstrated that apoptosis is induced in this transient assay system in the ab-sence of p35, we know that another antiapoptosis baculovirus gene, iap, which appears to function in a manner different from that of p35, can fully substitute for p35 in this assay (27, 51). Thus, the effect of harvest time probably correlates with the time required to induce apoptosis following cotransfection. The effects of p35 are more pronounced when the lef library is used to supply the lefs rather than the AcMNPV clone library, which supplies additional genes (Fig. 4A). Assuming that the effect of p35 in this assay system is to block cellular apoptosis, this result suggests that there may be additional genes in the AcMNPV genome that modulate the rate of induction or the effects of apoptosis.

Transient-expression assays have been an extremely power-ful tool for the identification of the baculovirus genes involved

TABLE 2. Average activity of reporter plasmids in the presence of different regions of the AcMNPV genomea

Clone(s) omitted

Relative activity (%)

pCAPCAT p6.9hc phcwt p10hc

All 1 1 5 1

None 100 100 100 100

IE15 72 136 74 45

IE15 and BC5 143 96 62 126

BC5, HL8, and HL5 127 122 50 120

HL5, ETL7, and PstH4 89 113 57 150

ETL7, PstH4, and PstH1 125 87 70 135

PstH1 and PstH5 125 58 91 113

PstH5 and HC9 134 63 2 18

PstH5 and HC9b _{107 64 254 75}

HC9 and HC10 72 49 87 161

HC10 and pXmaB 114 101 96 215

pXmaB and HK5 94 162 269 203

HK5 and IE15 142 107 89 152

a_{The genomic clone(s) removed from the set of clones representing the entire}

AcMNPV library is indicated. Transfections in the first row (All) contain only the reporter plasmid indicated at the top of each column. Those in the second row (None) contain the entire library of clones. The activity of each reporter plasmid with the entire library was set at 100%. Removal of genomic clones was accom-panied by the addition of lefs contained within the clones removed, as in-dicated in Table 1, except that pPstHIEco1.4 was used to supply lef-10; pNspAfl was used to supply the 39k gene. The values represent averages of two indepen-dent experiments.

b_{pXA-7 was added to supply vlf-1.}

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in late gene expression. The ability to test the activity of mu-tated forms of these lefs as well as vlf-1 in these assays will now provide a powerful means of exploring structufunction re-lationships.

ACKNOWLEDGMENTS

We thank Jeanne McLachlin for constructing pXAdBSA. This work was supported in part by Public Health Service grant AI23719 from the National Institute of Allergy and Infectious Dis-eases.

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