Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5 kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42 kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

Genome Characteristics of the Cyclophragma Undans Nucleopolyhedrovirus: A Distinct Species in Group I of Alphabaculovirus.

The Cyclophragma undans nucleopolyhedrovirus (CyunNPV), a potential pest control agent, was isolated from Cyclophragma undans (Lepidoptera: Lasiocampidae), an important forest pest. In the present study, we performed detailed genome analysis of CyunNPV and compared its genome to those of other Group I alphabaculoviruses. Sequencing of the CyunNPV genome using the Roche 454 sequencing system generated 142,900 bp with a G + C content of 45%. Genome analysis predicted a total of 147 hypothetical open reading frames comprising 38 baculoviral core genes, 24 lepidopteran baculovirus conserved genes, nine Group I Alphabaculovirus conserved genes, 71 common genes, and five genes that are unique to CyunNPV. In addition, the genome contains 13 homologous repeated sequences (hrs). Phylogenetic analysis groups CyunNPV under a distinct branch within clade "a" of Group I in the genus Alphabaculovirus. Unlike other members of Group I, CyunNPV harbors only nine of the 11 genes previously determined to be specific to Group I viruses. Furthermore, the CyunNPV lacks the tyrosine phosphatase gene and the ac30 gene. The CyunNPV F-like protein contains two insertions of continuous polar amino acids, one at the conventional fusion peptide and a second insertion at the pre-transmembrane domain. The insertions are likely to affect the fusion function and suggest an evolutionary process that led to inactivation of the F-like protein. The above findings imply that CyunNPV is a distinct species under Group I Alphabaculovirus.

Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248).

Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus. It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.

Transcriptional analysis of the putative glycosyltransferase gene (amv248) of the Amsacta moorei entomopoxvirus.

Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus, was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5' and 3' untranslated regions analysis revealed that transcription initiates at position -126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene's promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases -198 to -235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A)2TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.

Construction and Rescue of a Functional Synthetic Baculovirus.

Synthetic viruses provide a powerful platform to delve deeper into the nature and function of viruses as well as to engineer viruses with novel properties. So far, most synthetic viruses have been RNA viruses (<30 kb) and small DNA viruses, such as bacteriophage phiX174. Baculoviruses contain a large circular dsDNA genome of 80-180 kb and have been used as biocontrol agents and protein expression vectors. Here, we report on the first synthesis of a baculovirus based on the type species Autographa californica nucleopolyhedrovirus, AcMNPV, by a combination of PCR and transformation-associated recombination in yeast. The synthetic genome, designated AcMNPV-WIV-Syn1, is 145 299 bp comprising the complete genome of AcMNPV except for the hr4a locus that was replaced with an ∼11.5 kb cassette of bacterial and yeast artificial chromosomal elements and an egfp gene. Sf9 insect cells were transfected with AcMNPV-WIV-Syn1 DNA and progeny virus was examined by electron microscopy, and assayed in one-step growth curves and oral infectivity. The results conclusively showed that the rescued virus AcMNPV-WIV-Syn1 had structural and biological properties comparable to the parental virus. We validated a proof of concept that a bona fide baculovirus can be synthesized. The new platform allows manipulation at any or multiple loci and will facilitate future studies such as identifying the minimal baculovirus genome and construction of better expression vectors. This is the largest DNA virus synthesized so far, and its success is likely to be the impetus to stimulate the fields of other large DNA viruses such as herpesviruses and poxviruses.

The Host Specificities of Baculovirus per os Infectivity Factors.

Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host's midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses.

Genome-wide analysis of differential mRNA expression of Amsacta moorei entomopoxvirus, mediated by the gene encoding a viral protein kinase (AMV197).

Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.

The ha72 core gene of baculovirus is essential for budded virus production and occlusion-derived virus embedding, and amino acid K22 plays an important role in its function.

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.

Role of interactions between Autographa californica multiple nucleopolyhedrovirus procathepsin and chitinase chitin-binding or active-site domains in viral cathepsin processing.

The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.

Cell-dependent production of polyhedra and virion occlusion of Autographa californica multiple nucleopolyhedrovirus fp25k mutants in vitro and in vivo.

Members of the family Baculoviridae are insect-specific dsDNA viruses that have been used for biological control of insect pests in agriculture and forestry, as well as in research and pharmaceutical protein expression in insect cells and larvae. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the family Baculoviridae. During infection of AcMNPV in permissive cells, fp25k mutants are positively selected, leading to the formation of the few polyhedra (FP) phenotype with reduced yield of polyhedra and reduced virion occlusion efficiency, which leads to decreased oral infectivity for insects. Here we report that polyhedra of AcMNPV fp25k mutants produced from different insect cell lines and insects have differences in larval per os infectivity, and that these variations are due to different virion occlusion efficiencies in these cell lines and insects. Polyhedra of AcMNPV fp25k mutants produced from Sf cells (Sf21 and Sf9, derived from Spodoptera frugiperda) and S. frugiperda larvae had poorer virion occlusion efficiency than those from Hi5 cells (derived from Trichoplusia ni) and T. ni larvae, based on immunoblots, DNA isolation and larval oral infection analysis. AcMNPV fp25k mutants formed clusters of FP and many polyhedra (MP) in the fat body cells of both T. ni and S. frugiperda larvae. Transmission electron microscopy revealed that the nature of virion occlusion of AcMNPV fp25k mutants was dependent on the different cells of the T. ni fat body tissue. Taken together, these results indicate that the FP phenotype and virion occlusion efficiency of fp25k mutants are influenced by the host insect cells.

Selection and characterization of Autographa californica multiple nucleopolyhedrovirus DNA polymerase mutations.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.

Complete sequence, analysis and organization of the Orgyia leucostigma nucleopolyhedrovirus genome.

The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).

Immediate-early protein ME53 forms foci and colocalizes with GP64 and the major capsid protein VP39 at the cell membranes of Autographa californica multiple nucleopolyhedrovirus-infected cells.

me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid.

Interaction of Autographa californica multiple nucleopolyhedrovirus cathepsin protease progenitor (proV-CATH) with insect baculovirus chitinase as a mechanism for proV-CATH cellular retention.

The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA-proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA-green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA-proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA-proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.

A soluble form of P74 can act as a per os infectivity factor to the Autographa californica multiple nucleopolyhedrovirus.

The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica multicapsid nucleopolyhedrovirus P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in the ODV envelope by a C-terminal transmembrane anchor domain and is N-terminally exposed on the ODV surface. In the present study, a series of N-terminal and C-terminal truncation mutants of P74 were evaluated for their ability to rescue per os infectivity of the P74-null virus, AcLP4. It was discovered that a P74 truncation mutant lacking the C-terminal transmembrane anchor domain of P74 was able to rescue per os infection. This result shows that a soluble form of P74 retains per os infectivity factor function and suggests that P74 may be complexed with other proteins in the ODV envelope.

Expression of heterologous genes in the Amsacta moorei entomopoxvirus.

Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph promoter contains a conserved TAAATG motif, which serves as the site of initiation for both transcription and translation. Additional sequences downstream of the conserved motif have been shown to be involved in high-level expression of the sph gene. As a first step towards developing a protein expression vector based on the sph locus, four recombinant AMEV viruses expressing either gfp or lacZ were constructed. Both reporter genes were expressed under the control of the sph promoter containing the TAAATG motif. An additional 6 bp or 21 bp of sph coding region was included in three of the recombinants, to be expressed as an N-terminal fusion protein of GFP or LacZ. GFP and beta-galactosidase expression was observed at 2 days post-infection and continued throughout the observation period. The highest level of reporter gene expression was observed in the recombinant containing 21 bp from the sph coding region. These results indicate that sph locus of AMEV can be used successfully to express exogenous genes.

Autographa californica multiple nucleopolyhedrovirus me53 (ac140) is a nonessential gene required for efficient budded-virus production.

me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (AcDeltame53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcDeltame53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.

Autographa californica multiple nucleopolyhedrovirus and Choristoneura fumiferana multiple nucleopolyhedrovirus v-cath genes are expressed as pre-proenzymes.

Intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH), which lacks canonical targeting signals, are poorly understood. The cathepsins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and most other alphabaculovirus group I nucleopolyhedroviruses have well-conserved N-termini containing overlapping chymotrypsin-cleavage (Y(11)) and myristoylation (G(12)) motifs, which are suggestive of proteolytic signal-peptide cleavage to generate proV-CATH and subsequent acylation. To determine proteolytic N-terminal processing of V-CATH, haemagglutinin epitope-coding tags were fused to the 5' and/or 3' ends of AcMNPV and CfMNPV v-cath. Immunoblot analysis suggested that a small N-terminal peptide is cleaved for both viruses, indicating that v-cath is expressed as a pre-proenzyme. The two viral homologues undergo similar proteolytic processing, but have different glycosylation or other post-translational modifications. An AcMNPV V-CATH-DsRED fusion protein co-localized to the endoplasmic reticulum with an HDEL motif-containing green fluorescent protein. Based on these findings, pre-proV-CATH processing and trafficking mechanisms are postulated.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.