Baculovirus diversity and molecular biology.

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BACULOVIRUS DIVERSITY AND MOLECULAR BIOLOGY Gary W. Blissard and George F. Rohrmann Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon

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Review The Baculoviridae are a family of occluded DNA viruses pathogenic pre dominantly for holometabolous insects. Although interest in baculoviruses originally centered on their natural ability to control insect pest populations, they have recently been used as expression vectors for the production of proteins for medical research and biotechnology (50). This has led to the widespread use of baculoviruses in many laboratories and an increased inter est in their biology. Several reviews have focused on baculoviruses, including overviews of their general biology (38), the molecular nature of the Baculo viridae and their practical application as insecticides (39, 67), their molecular biology (25), their application as expression vectors (52, 65, 66, 74) , and the structure of polyhedrin (9 1 ). In this review we describe our current un derstanding of molecular aspects of the baculovirus infection cycle and discuss the genetic organization and diversity of baculovirus genomes.

General Baculovirus Properties Baculoviruses are a diverse group of large viruses with covalently closed, double-stranded DNA genomes of 88-153 kbp (11,94), which are pathogenic for invertebrates. While the vast majority of baculoviruses infect insects [baculovirus infections have been reported in over 600 species of insects, (67)], several have also been found in crustacea (2 1 , 1 04). Baculoviruses are primarily pathogens of insects of the order Lepidoptera, but they also infect Hymenoptera, Diptera, Coleoptera, and Trichoptera. Taxonomically, baculo VIruses are divided into three subgroups: A, B , and C. Subgroup A, the 127 0066-41 70/90/01 0 1 /0 1 27$02.00


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nuclear polyhedrosis viruses (NPVs) , have many virions occluded within single intranuclear crystals called polyhedra. Subgroup B , the granulosis viruses (GYs) , have only a single virion within each crystal or "granule. " Subgroup C , the nonoccluded baculoviruses (NOBs), have no occlusion body surrounding the virions. NPVs are also frequently subdivided by the extent of aggregation of their nucleocapsids within the envelope; some are present singly (SNPVs) whereas others are found as multiples (MNPVs). However, the aggregation of nucleocapsids within envelopes (MNPV, SNPV) does not appear to be phylogenetically significant (62a). The host ranges of the baculo virus subgroups differ significantly. Most notably, viruses in subgroup B (GVs) have been reported only from lepidopteran larvae. For the purposes of this review, we focus on the subgroup A baculoviruses, the NPVs, which have the widest ranges of hosts and have been studied most intensively because cell lines permissive for their replication are readily available.

Life Cycle In the environment, NPVs are commonly found on plant surfaces and in the soil. The virions are occluded within polyhedral-shaped occlusion bodies that surround and protect the infectious virions (Figures 1 , 2 , 3). The occlusion body is composed predominately of a 29-kd protein termed "polyhedrin;" occlusion bodies are commonly referred to as "polyhedra. " Polyhedra are very stable and can persist indefinitely in the environment. When subjected to high pH (> 1 0) such as that found in a lepidopteran midgut, however, polyhedra dissolve and release the infectious virions. A unique feature of the NPV life cycle is the production of two virion phenotypes: Those virions found within polyhedra are termed "polyhedra-derived virus" (PDV); the other form, found in the hemocoel of the infected host insect, is termed "budded virus" (BV) (Figures 1, 2). Historically, a number of terms have been used for the two virion phenotypes: The PDV phenotype has also been called "occluded virus" (OV). Alternative names for the BY phenotype include "nonoccluded virus" (NOV) and "extracellular virus" (ECV or EV). Autographa californica MNPY (AcMNPY) is the most intensively studied baculovirus. Granados & Lawler (40) examined the pathway of infection of AcMNPV in larvae of Trichoplusia ni. After rapid dissolution of polyhedra in the midgut, the released virions (of the PDV phenotype) enter the host cells by fusion of the virion envelope with microvilli of midgut epithelial cells. In the midgut cells, nucleocapsids are transported into the nucleus where they uncoat as early as 1 hr post infection (p. i), and the virus undergoes a primary round of replication with progeny nucleocapsids observed as early as 8 hr p.i. Cytopathic effects observed during this phase include an enlarged nucleus and a virogenic stroma within the nucleus. At 12 hr p.i. some progeny nucleocap sids begin to bud through the nuclear membrane. In the cytoplasm the


Figure 1

Electron micrographs of nuclear polyhedrosis virus polyhedra and the two virion

phenotypes. A . A portion of the nucleus (Nu) and cytoplasm (C) of an AcMNVP-infected lepidopteran cell

(Lymantria dispar) late in infection. Polyhedra (PH) that contain occluded virions are present and the electron dense polyhedron envelope (PE) that surrounds each occlusion body is indicated. Progeny nucleocapsids are observed at the periphery of a dense virogenic stroma (VS).

B. A group of rod-like nucleocapsids (NC) characteristic of baculoviruses are shown budding through the plasma membrane (PM) of the host cell. Virions produced by budding are of the budded virus (BV) phenotype. C. A virion of the budded virus (BV) phenotype is shown with the plasma membrane derived

envelope (E) and the peplomers (pep) indicated by arrows. The virion is from L. dispar MNPV, and the bar represents 0. 1 /Lm.

D. A virion of the polyhedra derived virus (PDV) phenotype is shown in the nucleus of the cell. The envelope (E), which is assembled de novo in the nucleus, is indicated by an arrow. The virio.n js from AcMNPV, and the bar represents 0.1 /Lm. (Electron micrographs courtesy of Dr. J . R . Adams.)

Virogenic Stroma

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Ingestion by Insect Host Solubilization

of polyhedra in the Midgut

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Typical cellular infection cycle of the nuclear polyhedrosis virus. Polyhedra are

ingested by a susceptible insect and solubilized by the high pH of the insect midgut. Virions of thc polyhedra derived virus (PDV) phenotype are released and enter midgut epithelial cells by fusion with microvilli. Nucleocapsids are transported to the nucleus where uncoating of the viral DNA occurs, followed by gene expression and viral DNA replication. Progeny nucleocapsids are observed assembling within and around a dense virogenic stroma. Some progeny nucleocapsids bud through the nuclear membrane and are transported to the plasma membrane but apparently lose the nuclear derived envelope in the cytoplasm. These nucleocapsids then bud through the cytoplasmic mcmbrane into the hemocoel acquiring the budded virus (BV) specific envelope that contains the virus-encoded envelope glycoprotein (gp64). These virions (of the BV phenotype) appear to be specialized for secondary infection of other host cells. A second group of progeny nUcleocapsids become enveloped within the nucleus by a de novo assembled envelope. These virions are subsequently occluded within polyhedrin protein that crystallizes around them. Maturation of the polyhedra includes the addition of a polyhedral envelope around the periphery of the forming occlusion bodies. Upon insect death and cell lysis, the polyhedra are released into the environment. The diagram was modified from ( 1 1 2) .


Baculovirus Phenotypes

BV specific Components Peplomers

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Virus DNA

Envelope glycoprotein (gp64) Virion envelope (derived from plasma membrane)

Figure 3

PDV specific Components

Common Virion Components

M II

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Capsid Protein (p39)

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Basic DNA Binding Protein (p6.9)

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Budded Virus (BV)

Polyhedral Envelope (PE) Protein (p32-34) Virion envelope (assembled in the nucleus) Polyhedrin(p29)

Polyhedra Derived Virus (PDV)

Baculovirus structural components. The two baculovirus virion phenotypes are shown diagramatically with shared and phenotype-specific components indicated. It has been suggested that the gp64 protein may be the major component of the peplomers on the BV. For the purposes of this diagram, the BV phenotype is represented by a virion with a single nUcleocapsid although multiple nucleocapsids are also sometimes observed in BV virions. The PDV phenotype is represented by a diagram of an MNPV.


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envelope acquired from the nuclear membrane is lost, and the nucleocapsid is subsequently transported to and buds through the plasma membrane. These virions of the "budded virus" (BY) phenotype may infect many cell types (fat body, muscle, tracheal matrix, hemocytes, epithelial) producing a second round of replication. In the infected cell, progeny nucleocapsids produced in the nucleus may have two fates (Figures 1 , 2): They may move out of the nucleus into the cytoplasm and bud through the plasma membrane (budded virus, BV) , or they may be enveloped de novo in the nucleus and later occluded within polyhedrin (polyhedra-derived virus, PDV). Typically occlu sion bodies are first observed at 24 hr p.i .. With many NPVs, occlusion of virions may not occur in midgut cells. In the nucleus of an AcMNPV-infected midgut cell, some nucleocapsids are enveloped as early as 1 2 hr p.i. , but these PDV virions are rarely occluded (40, 41), indicating that the lack of occlusion bodies in midgut cells is not due to the absence of PDV virions. The two NPV virion phenotypes differ in virion morphology, protein composition , tissue specificity, and mode of viral entry into host cells (Figures 1, 2, 3). Virions of the BV phenotype occur primarily as single nuc1eocapsids in a loose-fitting viral envelope, whereas virions of the PDV phenotype may have one or many nucleocapsids enveloped in single tight-fitting envelopes (47, 48, 1 05). In addition, the envelopes of the two virion phenotypes are apparently derived from different sources (Figure 1 , 2) and appear to exhibit a marked tissue specificity. PDV envelopes appear to be specialized for interaction with polyhedrin (Figures 2, 3) and for infection of the columnar epithelial cells of the insect midgut, while BV envelopes are specialized for interactions with cells and tissues within the insect hemocoel. PDV, which is released from occlusion bodies by dissolution in alkaline solutions or midgut fluids, is highly infectious to midgut epithelial cells but is much less infectious in the hemocoel. Conversely, the BY phenotype from hemolymph or infected cell cultures is highly infectious in the hemocoel and in cell culture (38, 1 19). In addition to differences in tissue specificity, the two virion phenotypes also differ in the primary mode of viral entry into host cells. Whereas the PDV phenotype enters midgut epithelial cells by fusion of the viral envelope with microvilli, the BV phenotype primarily enters cells by adsorptive endocytosis (Figure 2 ) ( 1 17). These different mechanisms of virus entry are probably related to the differences in the viral envelopes. The B V envelope, acquired when the nucleocapsid buds through the cytoplasmic membrane, contains a viral-encoded envelope glycoprotein (gp64) (Figure 1 , 2). The PDV en velope, which is not well characterized, is acquired de novo in the nucleus where the PDV -yirions are subsequently occluded in polyhedra (38, 41 , 1 03). PDV envelopes do not contain the gp64 envelope glycoprotein. That the two virion phenotypes have quite different envelopes (Figure 2, 3) is consistent with the observed differences in biological activities of the two phenotypes.

BACULOVIRUS MOLECULAR BIOLOGY

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Thus, although the two virion phenotypes are genetically identical ( 1 00) , the two phenotypes differ in (a) morphology and protein composition (9, 105), (b) source of virion envelopes (38 , 1 03) , and (c) relative infectivities to cultured insect cells and to different cell types in the insect (38 , 53, 1 1 5 , 1 1 9).


Regulation of Gene Expression and Viral Replication In the infected insect cell, the expression of viral genes and DNA replication are believed to occur in an ordered cascade of events in which each successive phase is dependent on the previous phase. This cascade model was originally suggested from studies of pulse-labeled infected cell specific proteins (ICSPs) ( 1 5 , 24, 68, 1 32) and studies utilizing inhibitors of protein synthesis (56). Analyses of viral mRNAs of many genes by northern hybridization, S I nuclease protection, and primer extension have subsequently identified tem poral classes of viral gene transcription. To date, most evidence suggests that the cascade of viral gene expression is regulated at the transcriptional level with gene products of one temporal class of baculovirus gene transactivating (directly or indirectly) transcription of the genes of the next temporal class (44; for review, see reference 3 1 ). Baculovirus gene expression is divided into two general phases, an early phase that precedes viral DNA replication and a late phase that occurs as or after viral DNA replication begins. The early phase is subdivided into two functionally defined stages (immediate early and delayed early) distinguished as follows: Immediate early (IE) genes are those genes that can be transcribed by uninfected insect cells and require no viral gene products for their expression. Delayed early (DE) genes, on the other hand , require other viral gene products for their transcription. Late genes are those genes that are first transcribed after or concurrently with the onset of viral DNA synthesis and are transcribed from the late baculovirus AlaTAAG promoter. Hyperexpressed late genes are distinguished from other late genes by the fact that mRNAs from most late genes decline at very late times post infection , whereas mRNA levels of hyperexpressed late genes remain high throughout the infection cycle. Known hyperexpressed late genes include polyhedrin and p I O. The presence of immediate early and delayed early genes was initially suggested by work using the protein synthesis inhibitor cyclohexamide and amino acid analogs (56). When a cyclohexamide block was applied from 1 to 8 hours p.i. , certain proteins were synthesized immediately upon release from the block. Other early proteins appeared only after a delay. More direct evidence for the two early phases and the cascade effect was provided by work using reporter gene constructs (44, 46). In the later experiments, the reporter gene, chloramphenicol acetyltransferase (CAT), was fused to the open reading frame of the AcMNPV 39K gene so that the CAT open reading frame was expressed under the control of the 39K promoter. In such a


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construct, the relative activity of the 39K promoter is assayed by measuring CAT enzyme activity in cells transfected with the plasmid construct. When the 39K-CAT plasmid was transfected alone into SpodopteraJrugiperda cells, the 39K promoter was not active. However if the same plasmid was co transfected along with baculovirus genomic DNA, the 39K promoter was active. The portion of the genome responsible for this "transactivating" effect was eventually identified and a single gene isolated. This transactivating gene (termed immediate early gene 1, IE-I) is transcribed in S. Jrugiperda cells independent of any other viral transcription or products. Thus, the relation ship between immediate early and delayed early genes was demonstrated by showing that the transcription of a delayed early gene (39K) is dependent on the gene product of a transactivating immediate early gene (IE-I ) ( 1 3, 44, 46). Recently, the 35K gene of AcMNPV was shown to be transcribed as an immediate early gene if the hr5 enhancer was present in cis, and that transcription was greatly increased in the presence of IE-I (79) . Thus, baculo virus enhancer sequences (discussed below) may also play a role in the regulatory cascade. Baculovirus immediate early and delayed early genes appear to have promoter elements similar to those from eukaryotic organisms, as would be expected [or promoters that must be recognized by the host cells. Since insect genes are regulated in a complex manner, many different promot er motifs may be used by baculovirus immediate early genes . Indeed, the immediate early gene, IE-I, is transcribed from two early promoters. An upstream promoter appears to function only very early (0-2 hr p.i.) and gives rise to a longer spliced transcript that declines after 2 hr p.i. ( 17). A down stream, apparently constitutive, early promoter produces a shorter unspliced transcript ( 1 7, 46). Several consensus sequences upstream of baculovirus early genes have been observed (9, 79) . For a number of early baculovirus genes (lE-l spliced, IE- I unspliced, 39K delayed early, and the gp64 en velope glycoprotein) transcription initiates within a tetra-nucleotide "CAGT" motif which is perfectly conserved (9). A putative TATA box is also con served in sequence and position (2 1-24 nt) upstream of this CAGT motif. Similarities also exist between a possible consensus repeat from the hr5 enhancer (TjATjA CGNGTR) and sequences upstream of several early genes (79). Enhancers are cis-acting DNA sequence elements that increase the rate of utilization of promoters by RNA polymerase and function relatively in dependent of their orientation or distance (up or downstream) from the promoter. Functional enhancer elements that increase the rate of transcription of early baculovirus genes have been identified in AcMNPV. The AcMNPV genome contains five regions termed homologous repeats (hrl -hr5), each with repeated sequences containing multiple EeoRI sites ( 1 8). The repeated sequences in each homologous repeat contain multiple copies of a conserved



135

26-bp palindrome with an EcoRl site at its core (43). I n transient assays, the hr5 element enhances the transcription of immediate early genes (79) and delayed early genes (45). In one case (79), cis-linked homologous repeats enhanced immediate early transcription by 1 2-20 fold in the absence of the IE-I gene product, but transcription was increased 1 000-3000 fold in the presence of the IE- I protein. This suggests that while some genes may be transcribed initially at minimal levels (immediate early transcription) they may be transcribed at much higher levels in the presence of other immediate early gene products such as IE-I (delayed early transcription). It has been speculated that the activity of the homologous repeat enhancers may be stimulated by the IE-I gene product (43, 45). While these enhancers may play an important role in early transcription of AcMNPY genes, it is not known if functionally similar enhancers are common to other baculoviruses (see be low). The late phase of viral expression is defined as the viral transcription that occurs after or concurrently with viral DNA replication. Late genes are not transcribed when cells have been previously treated with aphidicolin, an inhibitor of a virus-induced DNA polymerase activity (76, 1 2 1 ). All late genes identified to date appear to be transcribed from the consensus late promoter element, ATAAG or GTAAG. This core element appears to func tion as both promoter and mRNA start site (87, 9 1 ). Transcription start sites for late baculovirus genes are usually mapped within this ATAAG motif and usually to the T and/or A at positions 2 and 3. In several baculovirus genes , multiple copies of the ATAAG motif are located upstream of the open reading frame (ORF), and transcription may initiate from several or all upstream ATAAGs (8 , 9, 1 08). Because this very short sequence motif serves as the late promoter, it may be selected against in the open reading frames of baculovirus genes. The transcription of late baculovirus genes may result from a viral-encoded or modified host RNA polymerase that recognizes the ATAAG promoter. This short promoter motif at the transcription initiation site is unusual in structure when compared to typical promoters recognized by nuclear eukary otic RNA polymerases and bacterial RNA polymerases (64). Both bacterial and nuclear eucaryotic promoters usually consist of multiple noncontiguous blocks of sequence information. In contrast, the short promoter motif at the transcription start site of baculovirus late genes appears to be more similar to promoters recognized by RNA polymerases specific to yeast mitochondrial DNA and certain bacteriophages (T7 and T3) (7 , 69 , 95). These promoters also consist of short sequences located at the transcription start site. Baculo virus late gene promoters also share actual sequence similarities with the promoters from yeast mitochondrial DNA. On yeast mitochondrial DNA, transcription starts at the terminal nucleotide of highly conserved 9-nt promot-

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er sequence ATATAAGTA (7 , 95) , whereas transcription of baculovirus late genes usually starts within the highly conserved 5-nt promoter motif, A/GTAAG. There are several types of nuclear eukaryotic RNA polymerases. Each has specific functions and recognizes different types of promoters (64). RNA polymerase I, which is found in the nucleolus, is responsible for transcribing ribosomal RNA genes that account for 50-70% of cellular RNA synthesis RNA polymerase II is responsible for synthesis of most heterogenous nuclear RNA, the precursor to mRNA. RNA polymerase II accounts for 20-40% of the total RNA synthesis. RNA polymerase III activity in the cell is responsible for the synthesis of small nuclear RNAs (snRNAs) and tRNAs, and this enzyme accounts for less than 10% of total RNA synthesis. Eukaryotic RNA polymerases are also classified by their relative resistance to a fungal toxin, the bicyclic octapeptide alpha-amanitin. RNA polymerase II is extremely sensitive to inhibition by alpha-amanitin. RNA polymerase III from vertebrate cells is more resistant, and RNA polymerase Ills from insect and yeast cells are highly resistant, to inhibition by alpha-amanitin. RNA polymerase I is considered to be uninhibited by alpha-amanitin. Evidence for a unique RNA polymerase that recognizes the late baculovirus promoter includes the discov ery of an alpha amanitin-resistant RNA polymerase activity (in AcMNPV infected cells) which was shown to be distinct from host RNA polymerase (34, 42). This activity is probably associated with recognition of late baculo virus promoters (A/oTAAG). Since an alpha amanitin-resistant RNA polymerase activity is induced late in infection , and since baculovirus late gene promoters are dissimilar to nuclear promoters, it is unlikely that baculo virus late genes are transcribed by cellular RNA polymerase II. Thus, baculo virus late genes are probably transcribed from either a modified host RNA polymerase or a viral-coded RNA polymerase that may be similar to the RNA polymerase of yeast mitochondrial DNA or the T7 or T3 bacteriophage. Two types of baculovirus genes are routinely distinguished late in infection: late genes and hyperexpressed late genes. Late genes are those genes whose mRNAs are found in maximal abundance just after DNA replication and decrease thereafter. Hyperexpressed late genes are late genes whose mRNAs are present at very high levels shortly after DNA replication and throughout infection. While it is known that the mRNAs and proteins of hyperexpressed late genes accumulate to extremely high levels that are maintained throughout the infection cycle the specific reasons for this are unclear. Differences between the two classes of late genes (late and hyperexpressed late) may be related to (a) the rates of transcription initiation from promoters of the two classes, andlor (b) relative mRNA half lives. At present, the distinction between the two classes of late genes is not well defined. It is likely that in hyperexpressed late genes, a 1 2-nt consensus sequence (AATAAGTATTTT)


.

,



1 37

surrounding the late gene "core" (ATAAG) may be important in the elevated levels of the hyperexpressed mRNAs and proteins (91). The above temporal classification of baculovirus gene expression is likely an oversimplification; our understanding of baculovirus gene expression is based on detailed studies of only very few genes. Regulation of baculovirus gene expression almost certainly will prove to be much more subtle and diverse. Baculovirus replication appears to be accomplished via a viral-encoded DNA polymerase. Higher eukaryotes possess four different types of DNA polymerases (alpha, beta, gamma, and delta), which are specialized either in their function or their location in the cell (6 I, 78). Both the alpha and beta types are localized in the nucleus with alpha DNA polymerase involved in nuclear DNA replication and beta involved in DNA repair. DNA polymerase alpha is sensitive to the antibiotic aphidicolin. DNA polymerase gamma is present in both the nucleus and the mitochondria and is thought to be involved in the replication of mitochondrial DNA. DNA polymerase delta appears to be similar to DNA polymerase alpha both in its sensitivity to aphidicolin and in its nuclear localization, but DNA polymerase delta is serologically distinct, differs in sensitivity to a number of nucleotide analogs, and has a 3' to 5 ' exonuclease activity that alpha lacks. Also, DNA polymerase delta i s stimu lated by proliferative cell nuclear antigen (PCNA, also known as "cyelin") that does not affect DNA polymerase alpha. Early studies (54, 55, 76) noted that a new DNA polymerase activity was induced in baculovirus-infected cells late in infection and was subsequently characterized as having delta-like properties (8 1 ). Recently, a baculovirus early gene encoding a DNA polymerase was located and sequenced (109). In addition to this DNA polymerase, baculoviruses also possess a gene encoding a protein termed ET-L, which shows amino acid sequence similarity (42%) to rat PCNA (22, 8 1 ). In a mutant virus containing an inactivated ET-L gene, late gene expres sion and DNA replication are delayed. This suggests that baculoviruses may utilize a DNA polymerase delta-related replication system. Cellular PCNA expression and localization are associated with the S phase of the cell cycle in mammalian cells (the S phase is the period of DNA synthesis) (84); therefore, it was suggested (8 1) that the stimulatory properties of the viral PeNA-like protein on DNA polymerase may allow the establishment of an S-phase environment and thus allow viral DNA replication independent of the host cell in which DNA replication either is not occurring or is shut down.

Baculovirus Structural Proteins and Genes Baculoviruses display a complex protein profile with certain proteins apparently specific to polyhedra, PDV, and BV. Although our current know ledge is incomplete, the following section summarizes the proteins that are well-characterized baculovirus structural components.

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The crystalline matrix of the occlusion body is made up of a 29-kd protein termed polyhedrin (9 1 , 1 14) . Polyhedrin is the baculovirus protein that has received the most attention because it is highly expressed (hyperexpressed) and may comprise up to 18% or more of the total alkali-soluble protein present in infected insects (85). Polyhedrin is the most highly conserved baculovirus protein so far characterized; there is over 80% similarity between different lepidopteran NPVs, over 50% similarity between lepidopteran NPVs and GVs, and about 40% similarity between a hymenop teran polyhedrin and polyhedrins from leipidopteran NPVs and GVs (9 1 ) (M. Harris, personal communication). No relatedness was detected between an N-terminal sequence of a dipteran NPV polyhedrin and polyhedrins from several other NPVs and GVs. Because polyhedrin is the occlusion body matrix protein, its major function appears to be stabilizing the virions in the environment, providing the viral DNA with protection from the UV of sunlight, and perhaps also protecting structural components of the PDV. The ability of the virus to occlude and thus protect its virions and persist in the environment outside of the insect host may be a trait subject to intense selection pressure since several other insect virus groups (entomopox viruses and cytoplasmic polyhedrosis viruses) have evolved the ability to occlude their virions. Because of the high level of polyhedrin gene expression, foreign genes are often cloned behind the polyhedrin gene promoter in recombinant baculovirus expression vectors (83 , 102). Although polyhedrin appears to be the major component of the occlusion body several other proteins may be involved in its structure. PIO is a hyperexpressed protein associated with viral occlusion, although its role in the formation of occlusion bodies remains unclear (23, 37 , 59, 63, 86, 1 1 3, 1 24, 1 25) . It appears to be associated with an elaborate network of rope-like structures that encircle and penetrate the nucleus of infected cells (23 , 86). These structures appear to be associated with the assembly of polyhedra. Recombinant baculoviruses that contained mutant or deleted pIO genes pro duced polyhedra that lacked a properly associated polyhedron envelope (PE; Figure 1 ) and were fragile and readily disintegrated when subjected to physi cal stress ( 1 1 3 , 1 25). This is in contrast to normal polyhedra, which are not readily disrupted by physical means. This sensitivity to physical stress is believed to be due to the improper association of a polyhedron envelope surrounding the polyhedra. In these plO mutant viruses, the PE is formed in the nucleus but is not efficiently transported and attached to polyhedra ( 1 25) . This suggests that pIO plays a role in the transport and/or attachment of the PE to polyhedra. It is interesting that cells infected with the mutant virus also failed to lyse late in the infection. The polyhedron envelope is a membrane-like structure surrounding polyhedra in electron micrographs (Figure 1 ). In the laboratory, the poly-


OCCLUSION BODY PROTEINS

,



1 39

hedron envelope frequently remains intact after dissolution of polyhedra, trapping virions within its collapsed structure. In nature, proteases of the insect gut probably degrade the polyhedron envelope soon after ingestion of polyhedra. Associated with the polyhedron envelope is a protein termed the polyhedron envelope (PE) protein that varies in size from 32 kd (in Orgyia pseudotsugata MNPV, OpMNPY) to 36 kd (in AcMNPY). Although it was originally determined that the polyhedron envelope from an NPY of Heliothus virescens was composed of over 90% carbohydrate (77), the polyhedron envelope protein appears to be a major component of the envelope. Using a differential dissolution protocol on polyhedra from AcMNPY, Whitt & Man ning ( 1 23) showed that a 36-kd protein was associated with the polyhedron envelope and required a reducing environment for release. Because of the reportedly high concentration of carbohydrate present in the envelope, they suggested that this protein was connected by thiol linkages to the carbohydrate envelope. Subsequently, Gombart et al (35 , 36) observed a similar protein associated with polyhedra of OpMNPY and located the gene in both Op MNPY and AcMNPY genomes . The OpMNPY and AcMNPY PE proteins are 58% similar in amino acid sequence. COMMON VIRION STRUCTURAL PROTEINS Within the nucleocapsid, viral DNA is associated with a very basic histone-like DNA-binding protein , designated p6. 9, which is encoded by the virus (57 , 75 , 1 1 1 , 1 26-1 3 1 ). The p6. 9 protein is a small, very basic, arginine-rich (40% arginine) protein associated with viral DNA in nucleosome-like structures in infected cells. It has been proposed that p6.9 is involved in the condensation of viral DNA prior to or during packaging. It has been suggested that upon infection, the p6.9 protein (associated with viral DNA in the nucleocapsid) may be phos phorylated, causing the "de-condensation" of the packaged viral DNA ( 1 1 1 , 1 27 , 1 28). Indeed, phosphorylation of purified nucleocapsids appears to result in the release of viral DNA ( 1 28). The p6.9 gene appears to be transcribed at high levels late in infection, consistent with the production of nucleocapsids and virions . This protein appears to be highly conserved among baculoviruses; it displays 80% amino acid sequence conservation between AcMNPY and OpMNPY (R. Russell & Rohrmann, submitted). B aculovirus capsids are rod-shaped structures that are assembled in the nucleus. Empty capsids are often observed in the nuclei of infected cells. The genes encoding the major capsid protein of OpMNPY and AcMNPY have recently been located and sequenced (8 , 82, 108). The capsid proteins of OpMNPY and AcMNPY have predicted sizes of 35 1 and 347 a.a. ' s with predicted molecular weights of 39.5 and 39 kd, respectively. On Coomassie blue-stained SDS-PAGE profiles of OpMNPY PDY and BY virions, the p39 capsid protein is one of the most intensely staining bands. Use of a monoclon-

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al antibody (MAb) to the OpMNPY p39 capsid protein confirmed that the same p39 capsid protein was present in both virion phenotypes (PDY and BY) (82). Similar results were obtained using MAbs to th� AcMNPY capsid protein (89) and a PAb to an AcMNPY capsid protein fusion (l08). In addition, the PAb reacted with the single major protein from isolated empty capsids (108). Comparison of the predicted amino acid sequences of the capsid proteins of OpMNPV and AcMNPV demonstrates that the capsid proteins of the two NPVs are well conserved with a predicted amino acid sequence similarity of approximately 56% (8, 108). BUDDED VIRUS SPECIFIC PROTEINS Budded virions that circulate in the hemocoel of an infected insect are budded from the surface of infected cells and derive the virion envelope from the plasma membrane of the cell (Figures 1, 2). SDS-PAGE protein profiles of BV virions are more complex than PDY virions, perhaps due to the presence of cellular proteins obtained during the budding process, or a greater number of viral-encoded proteins. Although the protein compositions of the two virion phenotypes are not well characterized, a major component of BV virions is a 64-kd envelope glycoprotein (gp64) which is not detected in the PDV virions (9, 49, llS). The gp64 protein is found in the cytoplasm at early times post infection and moves to the plasma membrane where it is acquired by BV during the budding process (9 , 118). The gp64 protein is both phosophorylated and glycosylated, and it contains disulfide bridges (89, 116, 118). In the membrane, gp64 appears to be present as tetramers of the gp64 protein (89, 116). In other studies (49, 53 , 89, 117), it was determined that certain MAbs directed against the gp64 envelope glycoprotein are capable of some degree of neutralization of BY virions. At saturating levels of the AcY 1 MAb, infectivity of BY preparations is reduced by three to four orders of magnitude (53, 117). The observation that MAbs directed against the gp64 protein are capable of neutralizing infectivity of the BY (49, 89, 118) suggests that the gp64 protein plays a major role in the infection process. To examine the role of the gp64 protein in adsorption and penetration, the AcY 1 MAb was incubated with BY virions both before and after adsorbing virions to Spodoptera Jrugiperda cells ( 1 17). While this MAb had no apparent effect on adsorption, entry of virions into cells appeared to be inhibited. Studies using inhibitors of the endocytic pathway of viral infection indicate that baculoviruses, like influenza virus, primarily use the endocytic pathway for infection (117). In the endocytic pathway, the entire virion (including the envelope) is endocytosed into an intracellular vesicle (an endosome) , which then fuses with acidic vesicles in the cell cytoplasm. The low pH within the endosome is then believed to trigger fusion of the viral envelope with the endosomal membrane, releasing the viral nucleocapsid into the cytoplasm. Studies of BY entry into host cells in the presence of lipid



141

soluble bases (which inhibit the fusion of the viral envelope with the en dosomal membrane by elevating the pH) suggest that BY entry into cells is primarily by the endocytic pathway. Consequently it was hypothesized that gp64 may play a role in fusion of the viral envelope with the endosomal membrane (I 17). The neutralization of BY by AcYl is therefore probably due to a block in the ability of BY virions to use the endocytic pathway, at a stage after initial viral adsorption. The gene encoding this major envelope glycoprotein (gp64) was recently identified and sequenced in both OpMNPV and AcMNPV (9, 122). The OpMNPY and AcMNPY gp64 genes encode proteins of 509 and 5 1 1 amino acids, respectively, with an overall amino acid seq\lence similarity of 78%. The envelope glycoprotein contains two highly hydrophobic domains. The first 1 7-20 amino acids at the N-terminus of the envelope glycoprotein are hydrophobic and probably serve as the signal sequence for membrane inser tion. Computer predictions (9, 122) and limited N-terminal amino acid se quence data (99) indicate that this signal sequence is removed by cleavage between amino acids 17 and 18 in OpMNPY and 20 and 21 in AcMNPY. The hydrophobic domain located at the C-terminus probably serves as the trans membrane anchor sequence, holding the glycoprotein in the cellular mem brane and later the virion envelope. The N- and C-terminal regions are not highly conserved in amino acid sequence (40-59%), but the hydrophobic nature of these regions is conserved, and this probably relates to the predicted roles of these regions. The predicted N-terminal secretory signal sequence and C-terminal transmembrane anchor sequence are consistent with the dctection of the gp64 protein at the periphery of infected cells and on BY envelopes (9, 1 1 8) . Although the amino acid sequences of the terminal regions have di verged, maintaining the functionally hydrophobic nature, the internal regions of the two proteins are highly conserved, with an internal amino acid se quence similarity of approximately 83% . The positions of the potential N linked glycosylation sites are also conserved between the AcMNPY and OpMNPY proteins. Since this protein is a membrane glycoprotein, the highly conserved internal region of the protein would be the portion of the protein exposed on the surface of the virion.

Genome Organization and Baculovirus Genetic Relatedness Although early studies indicated that most baculoviruses display only limited genetic relatedness (5 1 , 10 1), recent detailed sequence comparison of sub stantial regions of the OpMNPY and AcMNPY genomes provides a clearer picture of both the striking conservation of some genes and the extensive diversity also present. The most well-characterized region (map units 80-90) begins with the gp64 gene and extends to the p74 gene (Figure 4c). At the left margin of this region lies the gp64 gene, which codes for the envelope


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BUSSARD & ROHRMANN

glycoprotein of the budded virus phenotype. The gp64 gene is highly con served (74% nucleotide and 7S% amino acid sequence similarities) and in AcMNPV is separated by 1 82 nt from a series of five conserved ORFs in the opposite orientation (Figure 4a,c) (81 , 1 22) . Although the genomic organiza tion of AcMNPV and OpMNPV are similar, this intergenic region in Op MNPV is approximately 1 320 bp and contains almost 1 kbp of intensely repeated DNA, over a third of which appears to be a repeat of the dinucleotide GT (9, 35). The five ORFs vary greatly in predicted amino acid sequence similarity (26-72%) . In both viruses this set of ORFs is transcribed in a similar manner, with a number of mRNA start sites initiating upstream of each ORF, and all the transcripts terminating at the same position downstream of ORF 5 . A difference in the OpMNPV and AcMNPV genomes occurs within the gene that encodes the polyhedron envelope (PE) protein (ORF 3; Figure 4c) (35 , 36, SI). In the AcMNPV gene, the predicted PE protein contains 1 6 ARG-SER repeats that are not present in the OpMNPV protein. Downstream of these 5 OFRs lies a region of major difference between OpMNPV and AcMNPV. In AcMNPV, a region of over 4 kbp separates ORF 5 and the p26 gene. Since ORF 5 and the p26 genes are separated by only 224 bp in OpMNPV, it appears that a major insertion of approximately 4 kbp of DNA has occurred in the AcMNPV genome. This insert contains the HindIII KlEcoRI-S/hr5 region of AcMNPV (Figure 4c). It was demonstrated by hybridization experiments that regions homologous to the AcMNPV HindIlI K and EcoRl-S fragments do not appear to exist in the OpMNPV genome (35 , 62) . The HindIII-KlEcoRl-S insert region in AcMNPV contains two actively transcribed ORFs encoding predicted proteins of 35 and 94 kd (32, 79), which may represent non-essential genes that confer a selective advan tage to the virus but are not found in other baculoviruses such as OpMNPV. Therefore, the large differences in genome sizes of occluded baculoviruses (88- 1 53 kb) (11, 94) may reflect the incorporation of genes from insects or other viruses and may possibly give certain viruses a selective advantage, while not being essential for their replication. Contiguous with the AcMNPV HindIII-KiEcoRI-S region is the homologous repeat sequence hr5 , which is also not present in the OpMNPV genome. In addition to the differences described above, two transposable elements have been observed in this region in certain strains of AcMNPV. These include an apparently nonautonomous transposable element that has inserted into ORFI of the 'Doerfler' strain of AcMNPV (35) and a retrotransposon observed in the 94-kd ORF in the AcMNPV HindIlI-K insert (33 , 73). Both homologous repeated sequences and transposable elements are discussed in more detail below.

Homologous Repeated Regions in Baculovirus Genomes The genome of AcMNPV has five regions that contain homologous repeated sequences termed hrl -5 (IS) (Figure 4a). As mentioned earlier, one of these


143

hr sequences (hr5) was demonstrated to function as an enhancer of transcrip tion of early genes in AcMNPY (43, 45, 79). Although the homologous

repeats may be important regulatory elements in AcMNPY, no evidence (by non stringent hybridization) exists for the presence of related sequences in the OpMNPV genome (6). Kuzio & Faulkner (58) and Arif & Doerfler (1) described the presence of DNA repeated in four regions of the Choristoneura fumiferana MNPV genome. However, hybridization data indicated that these


repeats were not related to the repeats of AcMNPV, and they lacked the

EcoRI sites characteristic of the homologous repeats of AcMNPY (58). Repeated sequences have also been reported in the Lymantria dispar MNPV (LdMNPV) genome (71) but have not been well-characterized.

Recombination in Baculovirus Genomes and Transposable Elements Recent studies suggest that transposable elements may play a major role in the

variation observed in baculoviruses. Transposable elements are responsible for a variety of genetic phenomena because of their capacity to cause in sertions, deletions, and other DNA rearrangements (for a review, see refer ence 97). Insertion of a transposable element within or near a gene can activate or alter the level, tissue specificity, or dev elopmenta l tim ing of gene expr ession (28, 72, 88). Excision of transposable elements can also influence gene expression. Excision is often imprecise, and this leads to sequence alterations at the excision sites (5, 10, 12, 20, 90, 93, 96, 106, 110). Such imprecise excision of transposable elements may result in proteins altered in size (98), enzymatic activities (26, 110), or developmental patterns of gene expression (19, 70). Therefore, the insertion and excision of transposable elements can be an important cause of genetic variability (for a review, see reference 107). Eukaryotic transposons often encode proteins required for their own transposition and excision (transposases), and the regulation of transposase activity is vital to the regulation of transposable element activity. For example, strain and tissue specificities of P factor transposition and excision are due to regulation of transposase activity (27, 60). S om e e uk ary ot

ic transposons (retrotransposons) are very similar in organization to the proviral forms of retroviruses and transpose through RNA intermediates. Other eukaryotic transposons, termed nonautonomous transposable elements, lack open read in g frames yet are capable of transposition when transposase activities are provided by another transposon. Most transposable elements described in baculoviruses appear to lack open reading frames and are prob ably nonautonomous (Table 1). Many transposons found in baculoviruses

contain 4 base pair (bp) direct repeats of the viral target sequence TTAA and flanking inverted repeat sequences of 10 to 32 nt on either end of the inserted DNA (2, 3, 14, 16, 29, 35, 120). This motif of inverted repeats flanked by direct repeats is characteristic of transposable elements found in both eukary-


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70% homology to the ends of a 1 . 6 kb transposon isolated from an FP mutant (FP 1 .6) of AcMNPV (Beames & Summers , submitted). Transposable elements have also been found at map units 2 and 46 in the AcMNPV genome ( 14). Transposable elements identified in baculovirus genomes appear to have originated from highly or moderately repeated host insect cell DNA (2 , 30 B eames and Summers, submitted). Although most of these transposable elements appear to lack open reading frames (Table 1 ) , it is evident they could have important influences on viral evolution. In the AcMNPV strain used by Oellig et al (80), the transposon contains the baculovirus late gene promoter sequence ATAAG (35). Oellig et al (8 1 ) report a transcript originating near this sequence within the transposon. The first ATG contained within such an mRNA could result in the translation of the 3 ' half of the ORF I polypeptide. In another case, the solo long terminal repeat remaining after the excision of a retrotransposon in AcMNPV, contained two bidirectionally oriented ATAAG sequences that cause the initiation of late transcripts in both directions (33). In the ORFs of baculovirus genes , the baculovirus late promoter motif (ATAAG) rarely occurs and therefore appears to be selected against. Howev er, this may not be the case in insect genes , which appear to have randomly distributed copies of the ATAAG motif. Therefore, in addition to disrupting viral genes , the insertion of transposable elements could also cause them to be expressed in an aberrant manner.


1 48

BLISSARD & ROHRMANN

In contrast to other FP mutants that appear to segregate around or within the 25k gene at 36.6-37.5 mu in AcMNPV, a copia-like transposable element (termed TE-D) of 7 . 3 kbp inserts at around 86.4-86. 6 mu and is also associated with an FP phenotype (73). Transposable elements such as copia and copia-like elements are flanked at both ends by long terminal repeats (LTRs) that encode the information necessary for transcription initiation and termination (64). The internal portions of such elements contain open reading frames resembling those of retroviruses, including homology to reverse tran scriptase. Copia elements are named for their "copious" transcription as large quantities of copia mRNAs are produced in the cell. Transcription initiates from the center of each of the terminal repeats, generating (a) transcripts that extend into the copia element and (b) transcripts that extend into adjacent genomic DNA. Thus copia elements also initiate transcription of genes downstream of the site of insertion. In the genome, copia elements are always intact. Individual LTRs are not left behind by copia removal as would be the case if the internal portion of the copia element were removed by recombina tion. The TE-D element of AcMNPV (33 , 73) is flanked by two LTRs (270 bp) that contain imperfect inverted repeats at their termini. The AcMNPV mutant virus carrying this TE-D element is termed FP-DL. The 7.3 kbp copia-like element in FPDL was classified as a middle repetitive sequence element with about 50 copies dispersed within the T.ni genome. The TE-D element in FP-DL inserts 384-nt downstream of the translation start of the 94k open reading frame (Figure 4b) (79) . Upon viral replication, the element is frequently excised (probably by recombination) leaving a single copy of the LTR at the original site of insertion (33). The resulting mutant, termed FP-DS , retains the FP phenotype. Studies of the FP-DS mutant (containing only the solo LTR) show that transcription in this region is disrupted by the bidirectional transcription initiated within the remaining LTR. Interestingly, the transcription from this LTR may not result from thc "promoter" signals utilized when the TE-D element is inserted in the insect genome but may result fortuitously from the fact that the solo LTR contains a palindrome of the core ATAAG baculovirus late promoter sequence. CTTATAAG GAATATTC Indeed, transcription from the solo LTR in FP-DS initiates at position 3 within the ATAAG motif. This and the temporal abundance of the resulting tran scripts are both consistent with transcription from promoters of late baculovi rus genes. Transcription of the TE-D element within the T. ni genome has not been investigated, so the nature of the insect promoter (if any) in the TE-D LTR is not known.


1 49


Summary and Conclusions With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and character ized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also , transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are sub sequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-KlEcoR I -S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination . In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly , the acquisition of species specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and B V. The definition of the regulatory mechanisms involved in the expres sion of these phenotypes is critical to understanding the biology of the Baculoviridae. Also of paramount importance is the understanding of factors

150

BUSSARD & ROHRMANN

that govern late gene expression and the hyperexpression of polyhedrin and p IO. We look forward soon to the determination of the complete sequence and analysis of the genomes of a number of baculoviruses. This information should allow the identification of the genes shared with other viruses and will form the basis for a more complete characterization of baculovirus genes, their functions , and the role baculovirus genome diversity plays in their interaction with insect populations.


ACKNOWLEDGMENTS The authors wish to thank Jean Adams for permission to use and for prepara tion of electron micrographs. We thank Rebecca Russell for providing un published data and Burton Beames, Malcolm Fraser, JaRue Manning , Lois Miller, Christian Okerblom , and Robert Possee for providing manuscripts prior to publication. We also thank M. Harris for providing unpublished sequence data, Eric Carstens and Albert Lu for providing unpublished data on the map locations of several baculovirus genes , and Just Vlak for permis sion to modify and use artwork. We thank Robert Granados and David Theil man for critical reviews of the manuscript. This work was supported by grants from the NIH (A 1 2 1 973) and the USDA (85-CRCR- 1 -2380). G. W. Blissard was supported by National Research Service Award AI l 07886-01 from NIH. Literature Cited I . Arif, B . , Doerfler, W. 1984. Identifica

tion and localization of reiterated sequ ences in the Choristoneura fumiferana MNPV genome. EMBO J. 3:525-29 2 . Beames, B . , Summers, M . D . 1988. Comparisons of host cell DNA in sertions and altered transcription at the site of insertions in few polyhedra bacu lovirus mutants. Virology 162:206-20 3 . Beames, B . , Summers, M . D . 1989.

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1 68:344-53 4. Deleted in proof 5 . Bender, W . , Akam, M . , Karch, F . , Beachy, P. A . , Peifer, M . , et al. 1983. Molecular genetics of the bithorax com plex in Drosophila melanogaster. Sci ence 2 2 1 : 23-29 6. B ickne l l , J. N. , Leisy, D. J. , Rohr mann, G. F . , Beaudreau, G. S. 1987. Comparison of the p26 gene region of two baculoviruses. Virology 1 6 1 :589-92 7. Biswas. T. K . , Edwards, J. c . , Rabino witz, M . , Getz, G. S. 1985 . Character-

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