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102

sid shells. Assembly of the nucleoprotein core may

be regulated by the state of phosphorylation of p6.9,

which modulates its affinity for the viral DNA (Oppen-

heimer & Volkman, 1995; Funk & Consigli, 1993).

The process of nucleocapsid maturation was shown to

require intranuclear actin (Charlton & Volkman, 1991).

In the presence of cytochalasin D packaging of viral

DNA into the capsid shells was disrupted by proteolyt-

ic degradation of the unphosphorylated form of p6.9

(Oppenheimer & Volkman, 1995). Consistent with this

model, there appear to be specialized regions within the

stromal matte which are revealed by partial extraction

of cells (Young et al., 1993) and by en bloc staining

with tannic acid and uranyl acetate (G.V. Williams,

unpublished observations). These regions are high-

ly enriched with DNAse-sensitive filament bundles

and were observed to be contiguous with the core of

forming nucleocapsids in adjacent intrastromal spaces

(Young et al., 1993). The sites are probably enriched

with newly synthesised genomes accumulating prior to

insertion into forming capsids.

DNA replication

Baculovirus DNA replication occurs within the

virogenic stroma and is dependant on a virally encod-

ed 126 kda DNA polymerase sensitive to aphidicolin

(Wang & Kelly, 1983; Tomalski et al., 1988). The

majority of the DNA replication machinery is consid-

ered to be virally encoded, although possible utiliza-

tion of some host cell components is not excluded.

Viral DNA synthesis starts 6–8 hr pi and continues for

at least 12 hours, declining in rate by 18 hr pi (Tjia

et al., 1979; Erlandson & Carstens, 1983). Few of

the accessory genes required for DNA replication have

been identified, although a non-essential proliferating

cell nuclear antigen (PCNA) homologue, normally a

component of DNA polymerase in eukaryotic cells,

was identified in the genome of AcMNPV (Crawford

& Miller, 1988; O’Reilly et al., 1989). Viral PCNA was

not essential to DNA replication but deletion of part of

the ORF caused a delay in all late viral gene expres-

sion, and slowed progression of infection in insect

larvae. Another viral gene, designated p143, has 7

regions of homology to known helicase proteins (Lu

& Carstens, 1992a), and a temperature sensitive lesion

within this gene caused a DNA replication defective

condition (Lu & Carstens, 1991). Recently a transient

complementation assay was used to identify essential

trans-acting factors required for plasmid DNA repli-

cation in insect cells. Cotransfection of cosmid clones

in a subtractive assay led to the identification of 6

genome regions encoding at least 7 genes essential to

viral DNA replication (Kool et al., 1994a). This assay

confirmed the absolute necessity of the p143 (helicase)

and p126 (DNA Pol) genes but not the vPCNA gene

for viral DNA replication. Further analysis indicated

that the viral genes encoding ie–1 and late expression

factors (lef) 1, 2, and 3 were also essential, and viral

DNA replication was stimulated by the p35, ie–2 (for-

merly ie-n), and pe38 proteins (Kool et al., 1994b).

The putative ORI (HR) sequences were not essential

for plasmid replication in the presence of transfected

cosmid clones (Kool et al., 1994a), but were essen-

tial in experiments that used whole virus as the source

of trans-acting factors (Kool et al., 1993). Viral DNA

replication normally occurs within the virogenic stro-

ma in baculovirus infected cells (Volkman & Keddie,

1990) and is concommitant with morphogenesis of the

structure (Knudson & Harrap, 1976), so the HRs may

be essential for efficient DNA replication within the

stroma. Comparison of NPV DNA replication proteins

with DNA replication proteins of Herpesviridae has

indicated possible homology between these virus fam-

ilies, and both seem to share a core set of 6 essential

genes. Based on this study, amino acid sequence align-

ments suggest that ie–1 codes for a single stranded

DNA binding protein, and lef 1–3 code for a primase-

associated protein, a DNA polymerase processivity

factor, and a primase, respectively (Kool et al., 1994b).

Late processes in morphogenesis

Virion phenotypes and biphasic replication

Intrastromal spaces communicate with the surrounding

nucleoplasm of the ring zone, hence mature nucleo-

capsids are able to migrate into this peristromal com-

partment. Nucleocapsids destined to be components of

BVegress from the nucleus at about 24 hr pi (Fraser,

1986; Kawamoto et al., 1977). Later, BV production

is curtailed by an undefined mechanism in favour of

intranuclear envelopment of nucleocapsids (Figure 1;

Volkman et al., 1976; Stoltz et al., 1973; Tanada &

Hess, 1976). Nucleocapsids of both phenotypes are

seen to align with and attach to membranes at the api-

cal (nipple) end of the capsid (Kawamoto et al., 1977;

Fraser, 1986). Several methods of nucleocapsid egress

from the nucleus have been suggested, including exit

via nuclear pores (Hess & Falcon, 1977; Summers,

1971), migration into or through the cellular endomem-

103

brane systm, or passage through discontinuities in the

nuclear membrane (Adams et al., 1977). Of interest

is the apparent loss of the nuclear membrane-derived

vesicle in transit, as naked capsids are predominant at

sites of budding along the plasma membrane. No mod-

el for the release of capsids from transport vesicles

has been proposed; the process may involve fusion or

disintegration of membranes. Cytoplasmic extrusion

of nuclear membrane vesicles is not strictly dependant

on the presence of capsids, as mutants defective in

late gene expression commonly induced budding of

the nuclear membrane independant of capsid forma-

tion (Carstens et al., 1994). Because some late pro-

tein synthesis did occur,some capsid components, in

particularthose proteins associated with the apical cap

strucuture, may have induced the extrusion phenom-

enon.

Glycosylation is an important process in the mor-

phogenesis of mature budded virions. Kelly & Lescott

(1983) reported that TniMNPV grown in Sf cells

required N-glycosylation for proper envelopment of

virions, and in the presence of tunicamycin nucleo-

capsids accumulated in the cytoplasm of drug-treated

cells and budding at the plasma membrane was reduced

or eliminated. A modest dose of tunicamycin

caused a 90% reduction in virus titre. In a similar

study in which AcMNPV replicated in Tni cells there

was again a decline inproduction of infectious virus

in the presence of tunicamycin (Stiles et

1983). Progeny virus was morphologically indis-

tinguishable from normal BV although they were of

lower buoyant density.

Switchover from BV to PDV phenotype morphogenesis

The intranuclear ring zone is site of several mor-

phogenic processes closely associated with OB devel-

opment late in infection, including intranuclear envel-

opment and occlusion of virions, condensation of poly-

hedrin into paracrystalline arrays and finally the attach-

ment of the occlusion body calyx. The mechanism of

switchover from BV to occluded virus is not known,

and although BV production is reduced substantially

in favour of intranuclear envelopment some budding

at the plasma membrane is observed late in infection

(48 hr pi). Concommitant with the decrease in BV pro-

duction, patches of membranous profiles and vesicles

develop within the nuclear ring zone which are pre-

cursor structures utilized for intranuclear envelopment

of virions. Based on the distinct dimensions and tril-

amellar morphology of the membranous profiles, and

apparent lack of continuity with the nuclear membrane,

these were postulated to be the result of de novo syn-

thesis (Stoltz et al

1973). Baculovirus infection has

been observed to induce an invagination of the inner

nuclear membrane (Tanada & Hess, 1976; Summers &

Arnott, 1969), and this may be the source material for

the intranuclear membrane vesicles.

Immunocytochemical localization studies have

indicated that a 66 kda structural protein associat-

ed with the PDV envelope (PDV-E66) co-localized to

these intranuclear membrane patches but not nuclear

membrane (Hong et al

1994). A 25 kda PDV enve-

lope protein of OpMNPV was found to localize to

virions within the ring zone and within mature OB

(Russell & Rohrmann, 1993), and was also identified

at the periphery of the virogenic stroma. The major

tegument glycoprotein exclusive to the occluded viri-

on phenotype (gp41) has a similar distribution (G.V.

Williams, unpublished observations), as does a capsid

end structure-associated protein (p78) found in both

viral phenotypes (Vialard & Richardson, 1993). The

most likely explanation for these observations is that

nucleocapsids leaving the virogenic stroma are inter-

cepted by molecules necessary for completion of mor-

phogenesis, and regulation of peri-stromal accumu-

lation might govern the switchover from budded to

occluded virion phenotypes.

OB morphogenesis

OB formation occurs by polyhedrin condensation with-

in the intranuclear ring zone of infected cells and is well

established by 24 hr pi (Volkman & Knudson, 1986).

The paracrystalline arrays are described as a face-

centered cubic lattice comprised of 4–5 nm subunits

packed in a rhombic arrangement (Federici, 1986; Kel-

ly, 1985; Harrap, 1972a). Virions within the matrix are

randomly distributed and do not interfere with the reg-

ularity of the lattice (Harrap, 1972a; Harrap & Payne,

1979). Harrap (1972a) examined polyhedrin crystals of

partially dissolved OB by TEM using a negative stain

procedure, and determined that polyhedrin oligomers

form a six nodal structure. The size and shape of OBs

are heritable features (Gershenson, 1960) and are inti-

mately related to the primary sequence of polyhedrin

(Brown et al

1980; Carstens et al

1986; Carstens et

1992). Alterations in the polyhedrin coding region

cause dramatic alterations in the form of the paracrys-

talline lattice (Brown et al

1980; Duncan & Faulkner,

1982; Carstens et al

1986, 1987, 1992) and usually

abrogate the occlusion of virions, but no clear peptide

104

domain or signal motif has been associated with virion

incorporation.

Polyhedrin (29 kda) is a virus encoded protein

having 245 amino acid residues (Summers & Smith,

1976; Rohrmann, 1986; Rohrmann, 1992); it possess-

es a nuclear localization motif (Jarvis et al., 1991)

which facilitates accumulation in the ring zone. Post-

translational modification of polyhedrin includes phos-

phorylation (Maruniak & Summers, 1981; Dobos &

Cochran, 1980), and possibly glycosylation (Kelly &

Lescott, 1983), but the functional significance of these

post-translational modifications is not known. Poly-

hedrin and another hyperexpressed late gene product,

p10, are detected at about 12 hr pi in AcMNPV infect-

ed cells (Rohel et al., 1983) and production continues

throughout the replication cycle. The peptide p10 is

not a structural component of virions or OB, but forms

large fibrillar masses which are intimately associated

with maturing OB (Vlak et al., 1988; Williams et al.,

1989); thus some p10 may copurify with the occlusions

(Vlak et al., 1981). Morphogenesis of fibrillar bodies,

occlusion bodies, and the OB calyx seems to occur

independantly, as deletion mutants which affect one

of the structures do not affect formation of the other

structures (van der Wilk, 1987; Williams et al., 1989;

Zuidema et al., 1989), nor do these same mutants seem

to affect the intranuclear envelopment of virions.

In addition to the occluded virions several organic

and inorganic factors may also be incorporated intothe

OB matrix. A viral enhancing factor (VEF) in GV

and NPV occlusions which acts on the host insect

peritrophic has been identified (Derksen & Granados,

1988). As well, OB derived from insects may contain a

significant amount of silicon (Stairs, 1968) which may

be incorporated into the lattice, but the distribution has

not been determined nor have tissue culture derived

OB been examined for inorganic components. There

are also several accounts of the presence of RNA in

purified OB (Faulkner, 1962; Aizawa & Tida, 1963;

Himeno et al., 1969) in approximately equal quantity

relative to viral DNA (Faulkner, 1962). These mole-

cules and inorganic components have not been local-

ized within the OB structure, and may be associat-

ed with embedded virions, dispersed throughout the

occlusion matrix, or copurified as contaminants at the

surface of the OB.

Regulatory signal(s) which govern cessation of

crystal growth and attachment of calyx is (are) not

known, and the process may be mediated in a temporal

fashion by specific enzymes or may be inherent to the

crystalline structure. At least one polyhedrin mutant

displayed greatly increased affinity for calyx precursor

(Carstens et al., 1992), suggesting that a conforma-

tional shift in the polyhedrin molecule or oligomeric

superstructures may be involved in this process.

Calyx morphogenesis and attachment

The calyx is an electron dense carbohydrate-rich struc-

ture (Minion et al., 1979; Whitt & Manning, 1988),

and has also been called the OB membrane (Mack-

innon et al., 1974; Harrap, 1972a) and OB envelope

(Longworth et al., 1972). A TEM study of the calyx

by negative staining indicated a regular array distinct

from the polyhedrin lattice, with 15 nm holes or hol-

lows evenly distributed across the structure (Harrap,

1972a). After release of PDV from occlusions by alka-

li dissolution the calyx remains as a residual bag-like

structure (Harrap, 1972a).

The ontogeny of the calyx is unknown and is con-

sidered a de novo virus structure which forms as bil-

amellar sheet precursors in association with intranu-

clear p10 fibrillar bodies (MacKinnon et al., 1974;

Van der Wilk et al., 1987). Calyx precursor formation

is not dependant on the presence of p10 (Williams et

al., 1989), but this association is important for proper

attachment to maturing OB, and deletion of the p10

gene adversely affects the stability of the OB to with-

stand physical stress (Williams et al., 1989). A 34 kda

phosphoprotein (pp34; Gombart et al., 1989) has been

immunolocalized to the calyx (Russell & Rohrmann,

1990a; van Lent et al., 1990) and is covalently asso-

ciated by thiol bonds (Whitt & Manning, 1988). A

subpopulation of polyhedrin is also thiol linked to the

calyx (Whitt & Manning, 1988) which may anchor this

structure to the surface of the OB, although the spe-

cific interactions between calyx, pp34, and polyhedrin

have not been analysed. Deletion mutants lacking the

pp34 protein fail to produce a calyx, and the OB prod-

uct has increased sensitivity to alkali (Zuidema et al.,

1989; Vlak et al., 1988). A 34.8 kda spheroidin-like

protein (SLP; Vialard et al., 1990) has also been local-

ized to the OB of AcMNPV and may be a component

of the calyx (Rohrmann, 1992), but its role in calyx

morphogenesis or structure is not known, and in OpM-

NPV the putative SLP does not seem calyx associated

(Rohrmann, 1992).

Few polyhedra (FP) mutants

Distinct from mutations within the polyhedrin coding

region, several mutants of AcMNPV have been charac-

105

terized which also exhibit OB production manifested

as the few polyhedra (FP) phenotype. FP mutants arise

with high frequency in serially passaged NPV in tissue

culture (Potter et al., 1976) and the resulting OB con-

tain few if any occluded virions (Potter et al., 1976;

Ramoska & Hink, 1974). The genetic defect is general-

ly attributed to loss of a PDV-associated 25 kda protein

(Fraser et al., 1983; Beames & Summers, 1989) and

results in the production of fewer than 10 OB/cell. The

p25 gene codes for an envelope protein of the PDV phe-

notype and appears to be essential for envelopment of

intranuclear virions (Harrison & Summers, in press).

The protein could be an important factor for initia-

tion of OB formation, perhaps by causing nucleation

of polyhedrin (Wood, 1980); alternately, completely

enveloped intranuclear virions may trigger OB forma-

tion. The p25 gene is not exclusively responsible for

the FP phenotype because FP mutants were are also

generated by alteration of an early gene, p94 (Miller &

Miller, 1982; Friesen & Miller, 1987).

Cytolysis and OB release

Late in AcMNPV and SeMNPV infection, after OB

production and maturation, a nuclear disintegration

event occurs which facilitates the release of OB

(Williams et al., 1989; van Oers et al., 1993). This

process is dependant on the presence of p10 (Williams

et al., 1989) and the function has been mapped between

amino acids 52–79 in the case of AcMNPV (van Oers

et al., 1993). The mechanism and other potential com-

ponents involved in cytolysis and OB release are not

known. It was originally reported that release occurred

in both Sf and Tni cell lines starting at about 60 hr pi

(Williams et al., 1989). Recently we observed that Sf

21 and Sf9 cells cultured in our laboratory no longer

support release although normal cytolysis and release

of OB was observed in Tni cells (G.V.Williams, unpub-

lished observations). Preliminary experiments have

ruled out the cell or virus passage history and we cur-

rently attribute the phenomenon to subtle alterations in

components of the cell culture media.

Conclusion

In vitro studies have contributed greatly to an under-

standing of viral cytopathology, molecular biology, and

pathogenesis. A model of the role of baculoviruses in a

host-parasite relationship is developing which reveals

the virus as gaining control of many aspects of host

cell biology including control of the cell replication

machinery (apoptotic response, macromolecular syn-

thesis), the cytoskeletal structure, the nuclear mem-

brane and intranuclear architecture. Baculovirus repli-

cation is a collection of independent but inter-related

processes which work within the framework of the

host cell, with the in vivo goal of maximizing produc-

tion of progeny virions. Further molecular dissection

of baculovirus replication should yield insight into the

processes and principles of viral and host regulatory

systems, perhaps facilitating development of new gen-

erations of high efficiency sub-viral expression vector

systems and the development of genetically improved

strains of virus safe for field use in ecologically based

pest management strategies.

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Address for correspondence: Peter Faulkner, Department of Micro-

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K7L

3N6.

Cytotechnology 20:

111–123, 1996.

111

Construction of baculovirus recombinants

Paul A. Kitts

CLONTECH Laboratories, Inc., 1020 East Meadow Circle, PaloAlto, CA 94303–4230, U.S.A.

Key words: baculovirus expression system, baculovirus expression vector, recombinant baculovirus

Abbreviations: AcMNPV – Autographa californica multinucleocapsid nuclear polyhedrosis virus; SDS – Sodium

dodecyl sulphate; PCR – polymerase chain reaction

Introduction

There are three main reasons for wishing to modify

baculovirus genomes. The most common arises from

the use of baculoviruses to express foreign genes. Bac-

ulovirus expression systems frequently give high level

expression of proteins with full biological activity. This

combination of highly desirable features is responsible

for the widespread popularity of baculovirus expres-

sion systems. Second, is the renewed interest in the

use of baculoviruses as biological insecticides. Wild-

type baculoviruses are highly specific insecticides with

no toxicity for non-target organisms and have been

used to control insect pests for many years (Grana-

dos & Federici, 1986). Recently, considerable effort

has been made to enhance the insecticidal potency of

baculoviruses by introducing foreign genes, such as

hormones or toxins, or making other modifications to

the viral genome (for reviews see: Wood & Granados,

1991; Bonning & Hammock, 1992). The third reason

is that the ability to introduce mutations and other mod-

ifications into a baculovirus genome provides a power-

ful tool for studying baculovirus biology. Studying the

complex interactions between baculoviruses and their

hosts, both at the cellular and organismal levels, may

provide valuable insights into other viral infections. In

addition, greater understanding of baculovirus biology

is vital for the continued improvement of baculovirus

expression systems and baculovirus insecticides.

The large size of baculoviral genomes, 80–200 kb,

makes it difficult to manipulate the viral DNA direct-

ly due to its sensitivity to shearing and to the paucity

of unique restriction sites. The original method for

introducing mutations or foreign genes into a bac-

ulovirus circumvented these difficulties using a two-

step approach; a segment of the viral DNA was cloned

into a plasmid vector, modified, and then homologous

recombination inside insect cells was used to transfer

the modification back into the viral genome (Smith et

1983; Pennock et al

1984). Many recombinant

viruses have been made using this method; howev-

er, it is inefficient and time consuming. As the need

for recombinant viruses grew, more efficient methods

were developed. This review will describe the major

methods that have been developed to construct recom-

binant baculoviruses and their relative advantages and

weaknesses (for an earlier review see Davies, 1994).

The various methods are grouped according to the host

in which the recombinant is generated: insect cells,

heterologous hosts, and in vitro. Most methods for

generating recombinants have been developed using

AcMNPV, therefore this review will concentrate on

this virus.

Generation of recombinant viruses in insect cells

Recombination between a transfer vector and circular

viral DNA

The classical method used to introduce a foreign gene

or mutation into a baculoviral genome uses an inter-

mediate “transfer vector” (Smith et al

1983; Pennock

et al

1984). Such vectors consist of a bacterial plas-

mid into which a segment of the viral DNA has been

cloned. This piece of viral DNA can be manipulat-

ed by standard molecular biology techniques to create

mutations or to introduce foreign genes. Plasmid DNA