The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.     

Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.     

The immediate-early protein IE0 of the Autographa californica nucleopolyhedrovirus is not essential for viral replication

The role of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein IE0 in the baculoviral infection is not clear. In this study, we constructed the recombinant virus vAcDeltaie0 null for ie0 expression by targeted mutagenesis replacing exon0 with the cat gene. We found that vAcDeltaie0 replicated efficiently in Spodoptera littoralis SL2 cells, which are poorly permissive for AcMNPV. In contrast, in Spodoptera frugiperda SF9 cells, which are fully permissive for AcMNPV, vAcDeltaie0 DNA replication and budded virus production were delayed. These results and recently published data (X. Dai et al., J. Virol. 78:9633-9644, 2004) indicate that ie0 is not essential for AcMNPV replication but enhances it in permissive SF9 cells.     

Functional analysis of the Autographa californica nucleopolyhedrovirus IAP1 and IAP2

The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in de-tail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs ex-pressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.     

Semipermissive Replication of a Nuclear Polyhedrosis Virus of Autographa californica in a Gypsy Moth Cell Line

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [(35)S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early alpha proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.     

AcMNPVP35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly hedrovirus,HaSNPV)诱导的Tn Hi5 细胞凋亡,并能辅助HaSNPV在Tn Hi5细胞中复制,产生具有感染能力的子代病毒。瞬时表达实验证明,在Tn Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn Hi5细胞中的复制;进一步构建超表达p35 的重组病毒:vHap35,发现vHap35能够抑制Tn Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子。电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV)。     

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.     

Role of early and late replication events in induction of apoptosis by baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion

Summary The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.     

Baculovirus infection induces a DNA damage response that is required for efficient viral replication

Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10- to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.     

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the anti-apoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVDelta35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDeltaP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDeltaP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDeltaP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDeltaP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.     

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.     

Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 3 (LEF-3) is an essential protein for DNA replication in transient assays. P143, a large DNA-binding protein with DNA-unwinding activity, is also essential for viral DNA replication in vivo. Both LEF-3 and P143 are found in the nucleus of AcMNPV-infected cells, but only LEF-3 localizes to the nucleus when expressed in transfected cells on its own from a plasmid expression vector. P143 requires LEF-3 as a transporter to enter the nucleus. To investigate the possibility that LEF-3 carries a nuclear localization signal domain, we constructed a series of LEF-3 deletion mutants and examined the intracellular localization of the products in plasmid-transfected cells. We discovered that the N-terminal 56 amino acid residues of LEF-3 were sufficient for nuclear localization and that this domain, when fused with either the green fluorescent protein reporter gene or P143, was able to direct these proteins to the nucleus. Transient DNA replication assays demonstrated that fusing the LEF-3 nuclear localization signal domain to P143 did not alter the function of P143 in supporting DNA replication but was not sufficient to substitute for whole LEF-3. These data show that although one role for LEF-3 during virus infection is to transport P143 to the nucleus, LEF-3 performs other essential replication functions once inside the nucleus.     

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.