I INTRODUCTION

1. The Baculovirus Expression Vector System

2. Baculovirus Infection Process

3. Genes Involved in Viral Transcription and Replication

4. Baculovirus Host Range and Choice of Cells

5. Regulation of the Polyhedrin Gene Promoter

6. Aims and Objectives

Introduction 2

1. The Baculovirus Expression Vector System ·

The Baculovirus Expression Vector System (BEVS) has emerged as the

system of choice for the expression of foreign genes (for reviews [see

Luckow, 1991; Jarvis and Summers, 1992; O'Reilly et a/., 1992; Sridhar

et a/., 1994). Baculoviruses are insect viruses that infect mainly

· lepidopteran larvae. The Autographa californica nuclear polyhedrosis

virus (AcNPV) is most commonly used for expression work. The basic

biology centered around polyhedrin (polh ) gene expression during the

infection process underlies the utility of these viruses as expression

vectors (Miller, 1981 ). The polh gene of this virus, non-essential for the

replication of the virus in cell culture (Smith et a/., 1983), is expressed

to enormous levels during the late phases of infection. Therefore, in

most expression vectors, the poI h gene is replaced with the

heterologous gene which. is transcribed under the control of the

powerful polh promoter.

There are several features of the baculovirus system that are

particularly advantageous. Because an eukaryotic environment is being

used for protein production, biologically active proteins can be easily

obtained. Post-translational modifications like signal cleavage,

proteolytic cleavage, N-glycosylation, 0-glycosylation, acylation,

· amidation, phosphorylation, prenylation, and carboxyrnethylation can be

easily carried out by insect cells (O'Reilly et a/., 1992). Recombinant

proteins are targeted to their natural locations in the cell while

proteins containing signal peptides are recognized and cleaved in the

right fashion. The efficiency of secretion can be further enhanced by. __ _

fusing the honey bee melittin signal peptide (Tessier et a/., 1991 ).

Hetero- and homo-oligomeric assemblies have been demonstrated for a

wide variety of proteins in cells infected with baculoviruses. Using a

---

Introduction 3

baculovirus quadruple expression vector, four blue-tongue virus

proteins have been co-expressed and shown to assemble into virus-like

particles in insect cells (Belyaev and Roy, 1993). Exceptionally high

levels of expression is, perhaps, the most distinguishing feature of this

system. The highest level reported represents more than 50% of the

total cellular protein corresponding to approximately 1 gram of protein

product per 1 o9 cells (per 1 liter culture). Most heterologous proteins,

however, are produced at levels ranging from 10-100 mg per 1 o9 cells.

The use of very late promoters has a clear advantage of minimum

selective pressure on the virus to mutate towards heterologous gene

deletion or inactivation. Since baculoviruses are propagated at 270C,

the system can be used to produce large quantities of active protein

encoded by temperature sensitive alleles of a gene (Reynisdottir et a/.,

1990). The nucleocapsids of baculoviruses can accommodate 100 kbp of

additional DNA or more. Baculovirus vectors are helper-virus

independent and, therefore, relatively simple to use. All these reasons·

cited above help us to understand why BEVS has grown to become the

most powerful and popular expression system. The immense popularity

of BEVS is evident from the fact that more than 500 genes from

viruses, bacteria, fungi, plants, and animals have already been

expressed in this system (O'Reilly et a/., 1992).

However, the extent of glycosylation of some heterologous gene

products, such as tissue plasminogen activator, which require extensive

N-glycosylation, appears to decline late in infection (Jarvis and

Summers, 1989). This is probably due to an apparent decline in the

function of the endoplasmic reticulum due to a decline in host protein

synthesis during the late phase of infection. Therefore, an

overexpressed heterologous protein may not be post translationally

modified very well in this system due to the decline in functioning and

Introduction 4

because of the short time available prior to cell death caused by the

lytic nature of the virus. This .. secretory load.. is a' result of the

inability of the cells to cope with the processing of a large excess of

protein synthesized under the transcriptional control of a very late

promoter (Sridhar et a/., 1993a; Sridhar et a/., 1994). The use of a

promoter activated earlier in the infection cycle solves this problem of

.. secretory load .. to a large extent (Sridhar, 1993; Sridhar et a/., 1993b;

Hasnain et a/., 1994; Sridhar et a/., 1994).

The method of selection for recombinant viruses has evolved to a

great degree over the last few years. Traditionally, recombinants were

generated by in vivo recombination at the polh locus - two point

cross-overs between the viral DNA and a transfer vector carrying the

foreign gene flanked by viral sequences resulting in recombinant

progeny viruses having a replacement of the polh gene with the foreign

gene. These recombinants, however, appear at a very low frequency

(0.1-1 %) making microscopic screening (for polyhedrin negative

plaques) a very tedious process. Alternative methods to facilitate

screening include use of co-expressed reporter genes such as lacZ

(Weyer et a/., 1990) and luciferase (Hasnain et a/., 1994), antibody

screening, and PCR screening (O.Reilly et a/., 1992). Recently, a highly

efficient method which gives extraordinarily high rates of

recombination (>95%) has been reported (Kitts and Possee, 1993). Here,

an essential gene function adjacent to the polh locus, corresponding to

ORF 1629, is deleted from the wild type virus used for co-transfection.

This gene function can be complemented only after recombination with

a transfer vector that carries the foreign gene along with the deleted

complement of the viral gene thereby making it virtually impossible for

wild type progeny viruses to appear.

Introduction 5

2. Baculovirus Infection Process

AcNPV has a double-stranded, covalently closed, and circular DNA

genome of 128 kbp which has been completely sequenced (Ayres et a/.,

1994). The DNA is condensed into a nucleoprotein core which is present

within an enveloped capsid. These nucleocapsids are made in the

nucleus of infected cells. Two biochemically and morphologically

distinct progeny virus forms are produced during the infection process:

i) Budded viruses (BV), which are released into the extra cellular fluid

with a loosely fitting membrane envelope, and ii) Occluded virus (OV),

consisting of enveloped nucleocapsids embedded in a crystalline matrix

composed mainly of a protein called polyhedrin (Harrap, 1972). The

polyhedrin matrix protects the virus particles from inactivation by

physical factors after they are released into the environment.

Progress of infection in the insect: Insect larvae get infected

when they ingest OVs as contaminants of their food. The polyhedrin

matrix is solubilized in the midgut of the insects, releasing the virions

which enter midgut cells by fusion with the membrane of microvilli

(Granados and Williams, 1986). Infection of the polarized midgut cells

results in BV release from the basement membrane side of the cell

(Keddie et a/., 1989). This BV can gain access to the hemocoel and is

transported via the hemolymph to other tissues. BV released from the

midgut cells also infect epithelial cells of tracheoles, which provide

oxygen to the midgut, spreading infection along the tracheal network.

OVs produced by the infected tissues spread the infection to other

larvae after disintegration of the host.

Progress of Infection in Cell Culture: For repli.cation in cell

culture, the infection cycle occurs in three basic phases: early, late,

Introduction 6

and very late.

Early Phase - During this phase (approximately the first 6 hours

of infection) the cell is reprogrammed for virus replication. Infection in

cell culture is mediated by BVs entering by adsorptive endocytosis.

Nucleocapsids migrate through the cytoplasm into the nucleus where

the nucleoprotein core is released (Granados and Williams, 1986). There

is no requirement of viral protein synthesis for early gene transcription

(Nissen and Friesen, 1989). However, many early promoters are

transactivated by the product of the viral /E-1 gene (Guarino and

Summers, 1986) which is believed to enter the cell as a component of

the virions (Miller, 1988). Early gene transcription is thought to be

mediated by hos.t RNA polymerase II because no viral gene expression is

required and early gene transcription is sensitive to a-amanitin (Grula

et a/., 1981; Huh and Weaver, 1990).

Late phase - The late phase extends from 6 h pi (hours post

infection) to approximately 20 h pi. This is a period of extensive viral

DNA replication, late gene expression, and BV production. The A eN PV

genome has six . interspersed homologous regions, designated hr 1 to h r

5 (hr 4 contains two distinct hrs, designated hr 4a and hr 4b), of

approximately 500-800 bp in length that are believed to function as

origins of replication (Kool et a/., 1993).

Very late phase - This phase begins around 20 h pi.The polh

gene is· tiypertranscribed during this phase and the enveloped

nucleocapsids are embodied within the polyhedrin matrix of occlusion

bodies. Lysis of infected cells begins about 60 h pi, and by 72 h pi most

cells are in the process of dying and/or lysing.

Late and very late viral gene transcription, however,.· is

insensitive to a-amanitin (Grula et a/., 1981; Huh and Weaver, 1990).

Biochemical evidence suggests· that a new RNA polymerase is present in

Introduction 7

virus-infected cells which could be a virus-modified host RNA

polymerase, a new RNA polymerase with virus-encoded subunits, or

some combination of these two possibilities (Fuchs et a/., 1983; Yang e t

a/., 1991 ). Late and very late transcription is dependent on early viral

gene expression and on DNA replication -both cycloheximide and

aphidicolin block transcription of late and very late genes (Rice and

Miller, 1986).

Virus effects on host gene expression: AcNPV infection results in

shut-off of host gene expression, though host chromatin structure

appears to remain largely intact. A decline in steady-state levels of

host mRNAs begins approximately 12 h pi. By 24 h pi, steady-state

levels of cellular mRNAs such as actin and histone are quite low (Ooi

and Miller, 1988). Decline in host protein synthesis starts by 18 h pi,

and shut-off is virtually complete by 24 h pi (Carstens et a/., 1979). By

24 h pi, gene expression is primarily, if not exclusively, viral-specific.

The mechanism(s) by which host RNA and protein levels are down­

regulated are not known. The virus encodes a protein (p35) that may be

responsible for shutting off cellular apoptotic gene function(s) thereby

enabling the virus to complete its own life cycle ending in lysis of the

host cells (Sugimoto et a/., 1994).

3. Genes Involved in Viral Transcription and Replication

Transcription regulatory genes: Baculovirus genes are transcribed

in a regulated cascade corresponding to the a, p, y, and o temporal

stages. The ·gene products of one temporal class activate, directly or

indirectly, the products of the next temporal class. Several genes that

appear to be involved in gene regulation have been identified. These

Introduction 8

genes include those encoding IE-0, IE-1, lE-N, PE-38, and CG30 (O'Reilly

et a/., 1992). IE-0 is actually an exon of one of the two forms of IE-1.

IE-1 is able to transactivate some early promoters in transient

expression assays (Guarino and Summers, 1986). The other three

proteins, lE-N, PE-38, and CG30, all share common unique structural

motifs: an unusual, double zinc-fingerlike motif and a C-terminal

leucine zipper. The unusual zinc-fingerlike motif is found in other

proteins, which are thought to have DNA-related functions (Free mont e t

a/., 1991 ). Thus, baculoviruses appear to have a family of related genes

that are probably involved in gene regulation.

Genes involved in DNA replication: Genes that are involved in viral

DNA replication include dnapo/, he/, and pcna. Enzymatic studies suggest

that not only is a new DNA polymerase activity induced during infection

but a host DNA polymerase activity is also stimulated (Miller et a/.,

1981 ). The dnapol gene (Tomalski et a/., 1988) encodes a 114-kDa

protein with homology to other DNA polymerases (eg., DNA polymerases

of herpes viruses, poxviruses, and adenoviruses). It is likely that this

gene encodes the AcNPV-induced, aphidicolin-sensitive DNA polymerase

described by Miller et a/. (1981 ). PCNA (proliferating-cell nuclear

antigen) may serve as a processivity factor for one of the DNA

polymerases. The he/ gene codes for a 143-kDa protein that contains a

region sharing sequence homology with other proteins having ATP­

dependent helicase activity (Lu and Carstens, 1991 ).

4. Baculovirus Host Range and Choice of cells

Baculovirus Host Range: Baculoviruses have a narrow host range

with each baculovirus being able to infect only a few taxonomically

Introduction 9

related insect species (Groner, 1986). In a controlled survey done by

Bishop et a/. (1988) it was seen that A cNPV, which is considered to

have a relatively broad host range for a baculovirus, nevertheless, has a

highly restricted host range. A cNPV can infect about 40 different

Lepidopteran species belonging to 11 families and it can also

additionally infect a Coleopteran cell line (Croizier et a/., 1994). The

silkworm Bombyx mori is not the natural host for AcNPV. However,

Morris and Miller (1993) have reported .. non-productive., infections of

Bombyx mori cells with A cNPV i.e. there is no evident polyhedra

formation though there are low levels of late gene expression. They

conclude that .. non-productive.. infections in non-permissive cell lines

are abrogated in a number of different ways and no one common

restriction point preventing productive infection is evident. A 79

nucleotide region within the baculovirus p143 helicase gene has been

identified to play an important role in conferring species specificity

(Croizier et a/., 1994). A hybrid virus which can infect both Bombyx

mori and Spodoptera frugiperda cells has been constructed by allowing

homologous recombination between AcNPV and BmNPV (Mori et at.,

1992).

According to studies conducted by Carbonell et a/. (1985) the

restriction to AcNPV infection in many non-permissive insects is not

the inability to enter those host cells but its inability to replicate its

DNA and express late gene products. This is confirmed by a study of

factors blocking late gene transcription (Rice and Miller, 1986).

However, there is almost no literature available on experiments carried

out to delimit factors controlling the ability of different insect cell

lines to support AcNPV DNA replication and late gene transcription.

Choice of Cells: The cells most commonly used with A eN PV -based

vectors are Sf9 and Sf21 cells. Both these cell lines are derived from

Introduction 1 0

pupal ovarian tissue of Spodoptera frugiperda (the fall armyworm),

(Vaughn et a/., 1977). Sf9 is a clonal isolate of Sf21. Other cell lines

that support replication of AcNPV are available. TN368 is a cell line

derived from minced adult ovaries of Trichoplusia ni (the cabbage

lo,oper) and this cell line is also used for expression vector work (Hink,

1970). Two cell lines, BmN and Bm5, derived from Bombyx mori or the

common silk worm are commonly used for infection with the Bombyx

mori nuclear polyhedrosis virus (BmNPV), the other baculovirus which

is also used for expression work. BmN and Bm5 are derived from pupal

ovarian cells (O'Reilly et a/., 1992).

5. Regulation of the Polyhedrin Gene Promoter

The polh promoter has been the "workhorse" promoter of BEVS and. is

one of the strongest promoters to be used in any expression vector

system.

Unusual structure of the polyhedrin promoter: The po/h promoter

of AcNPV has been extensively characterized by deletion and linker­

scan mutation analyses (Matsuura et a/., 1987; Possee and Howard,

1987; Ooi et a/., 1989). The transcription start point (Fig. 1) is at -50

relative to the A of the ATG translation start site (designated as +1 )

and lies within a highly conserved octanucleotide motif TAAGTATT.

This motif has been shown to be absolutely essential for transcription

initiation and the promoter has been defined as a 69 bp str~tch from -1

to -69. The sequence of the polh promoter is well conserved between

· otherwise distantly related baculoviruses (Rohrmann, 1986).

Mostly, bacterial and nuclear eukaryotic promoters have multiple

non-contiguous blocks of transcriptionally-important sequences

Introduction

-70 -50 +1

-----AATAAA I TAAGTATTI-1----------ATG

FIG. 1: Schematic representation of the AcNPV polyhedrln gene promoter. The essential polh promoter extends from -1 to -69 (the translation start point is designated as +1). The transcription start point. marked with a bent arrow, is at -50 and lies within a conserved, transcriptionally important TAAGTATT motif (boxed). The PPBP-cognate motifs (Burma et al., 1994) - AATAAA & TAAGTATT - have been shown. The 18 bp 'minimal' promoter (Morris and Miller, 1994) has been highlighted by a hatched bar.

Introduction 1 1

present upstream or downstream to the transcription start point. In

contrast, the polh promoter has an unusual structure and is similar only

to certain yeast mitochondrial and bacteriophage T7 and T3 promoters

where there is a conserved, transcriptionally important motif at the

transcription start point (Masters et a/., 1987}. Also, other

determinants of promoter activity are present in the untranslated

mRNA leader region (Ooi et a/., 1989) which is another unusual feature

of this promoter.

Recently, Morris and Miller (1994) have shown that. an 18 bp

sequence surrounding the transcription start point (Fig. 1) is sufficient

for minimal promoter activity, whereas the sequences encoding the

untranslated mRNA leader are required for the very-late burst of

expression. The structure of the polh promoter is, therefore, similar to

that of certain eukaryotic TAT A-less promoters where the 'initiator'

(the sequence around the transcription start site) acts as a 'minimal'

promoter capable of directing basal levels of transcription (Smale and

Baltimore, 1989; Weis and Reinberg, 1992).

The LEF genes and polyhedrin promoter activity: Seven early

genes, designated /ef-1, /ef-2, lef-3, lef-4, /ef-5, /ef-6, and /ef-7 (for

late expression factor) have been shown to be essential but not

sufficient for late gene expression. (Li et a/., 1993; Passarelli and

Miller, 1993a, b, c; Morris et a/., 1994; Passarelli and Miller, 1994}. The

level (replication, transcription, or translation) at which these genes

act is, however, not known. In fact, Kool et a/. (1994) have suggested

that /ef-1 and /ef-2 may be involved directly in DNA replication and,

therefore, indirectly in polh gene transcription.

hr1 enhances transcription from the polyhedrin promoter: The

AcNPV homologous region hr 1, located -3.7 kb upstream of the po/h

Introduction 12

promoter, has been shown to enhance transcription of a luciferase

reporter placed under the control of the polh promoter in transient

expression assays using AcNPV-infected Sf9 cells (Habib et a/., 1995).

This enhancer function of hr 1 has been shown to be independent of its

putative role as a origin of replication ( ori ) - Dpn I sensitivity assays

for DNA replication show that enhancement is not due to an increase in

plasmid copy number due to the ori function of hr 1. Habib et a/.,

therefore, suggested that the two functions of hr 1 (ori and enhancer)

may be carried out by different sequence elements within the hr 1

region.

A putative negative regulator binding immediately upstream

to the polyhedrin promoter: Etkin et a/. (1994) have reported the

identification of a 200-kDa host factor binding specifically to the -72

to -86 region of the polh promoter of AcNPV. This binding activity was

found in uninfected cells and also in cells during early stages of viral

infection, but decreased by 18 h p.i. Preliminary experiments suggested

that this protein functions as a negative regulator and the virus may

utilize this factor to control the differential expression of late and

very late genes.

PPBP - An unusual factor interacting with the polyhedrin

promoter: Gel retardation assays using nuclear extracts from A eN PV­

infected Sf21 cell line revealed a 30-kDa host factor (polyhedrin

promoter-binding protein or PPBP) that binds to the po/h promoter

(Burma eta/., 1994}. A hexanucleotide sequence (AATAAA) within the

promoter is important for binding in association with the

transcriptionally essential TAAGTA TT motif (Fig. 1 ). PPBP has been

affinity purified to homogeneity and appears to be an unusual DNA­

binding protein with respect to its stability (binds between 0.2 to 2 M

Introduction 1 3

NaCI and at temperatures as high as 650C), high binding affinity (binds

even in the absence of non-specific DNA with an apparent dissociation

constant of -3.7 x 1 o-12 M) and high specificity of binding (binds in the

presence of a 50,000-fold excess of non-specific DNA). Phosphorylation

of this factor is essential for its DNA-binding activity. Interestingly,

the PPBP-cognate region corresponds to the minimal promoter

described by Morris and Miller (1994), (Fig. 1 ). The correlation between

functional promoter sequences and the factor-binding site argues that

the interaction of PPBP with the polh promoter must be an important

event in the activation of transcription from this promoter. It has been

proposed that PPBP might function in a manner analogous to the TATA­

binding protein in recruiting the virus-specific RNA polymerase and/or

basal transcription factor(s) to the polh promoter.

6. Aims and Objectives

The level of synthesis of heterologous proteins in BEVS varies

considerably and is thought to be due to a number of factors (O'Reilly e t

a/., 1992; Hasnain et a/., 1994; Sridhar et a/., 1994; Ranjan and Hasnain,

1995). Efforts to increase the level of expression of foreign genes in

baculovirus systems have been directed at optimizing the locations of

foreign genes in relation to transcriptional and translational signals

within the polh gene. There has been very little attention directed at

the influence of the host cell line on protein expression. Significant

levels of cell line-dependent differences in reporter gene expression

have been observed in a study carried out by Hink et a/. (1991 ). However,

the molecular mechanism(s) governing such cell line-dependent

differences in expression have not been worked out. The number of

insect cells which support productive AcNPV infections is very limited

Introduction 14

(Morris and Miller, 1992). Characterization of the nature of the block(s)

to productive AcNPV infection in refractive insect cells may provide

insights into baculovirus-host cell interactions and host range

determination. Understanding the molecular mechanism(s) that

determine the selectivity of baculovirus replication at the cellular

level would facilitate the assessment of recombinant baculovirus

safety to non-target organisms and might allow control of the host

range of these viruses. In order to address these questions we used a

recombinant baculovirus vAcBhCG-Iuc that carries the genes for BhCG

and firefly luciferase cloned under two independent copies of the polh

promoter (Jha et a/., 1992; Hasnain et a/., 1994). The expression of

these two reporter proteins was studied in a panel of insect cell lines

and studies were carried out to determine the molecular mechanism(s)

governing differential expression and blocks to productive infections.

As a first step, evidences were collected to determine the level at

which such cell line-dependent expression of foreign genes may be

affected.

I nspite of the fact that this system is widely used for

heterologous expression, very little is known about the regulation of

the polh promoter and the mechanism responsible for hyper­

transcription from this promoter. The only factor (PPBP) directly

interacting with the polh promoter has been recently identified and

characterized (Burma et a/., 1994). Given the importance of this unusual

factor,. the interaction of PPBP with the polh promoter in permissive

as well as non-permissive cell lines and with respect to differential

expression of foreign genes has not been worked out. We, therefore,

carried out experiments to study the interaction of PPBP with the polh

promoter in permissive and non-permissive cell lines and the

differences, if any, in terms of such interactions between the over and

Introduction 1 5

under-expressed cell lines.

The unusual structure of the polh promoter (with the 'initiator'

acting as the 'minimal' promoter) and the binding of PPBP at the

transcription start point poses the problem of whether binding would be

maintained even after melting of DNA at this point during transcription

initiation. We, therefore, carried out experiments to · investigate

whether PPBP exhibits any single-stranded DNA-binding activity. Such

an activity would allow PPBP to maintain its position when the DNA

helix melts during th~ initiation of transcription.

The objectives of the study described in this thesis, therefore, can be

summarized as follows:

1 ) To study levels of f3hCG and luciferase expression in a panel of

insect cell lines infected with a dual recombinant virus vAcBhCG­

/uc.

2) To study molecular mechanism(s) governing cell line ... dependent

differences in expression with respect to:

a) Virus entry

b) Virus replication

c) Transcription of reporter genes

d) Reporter mANA stability

3) To study DNA-protein interactions at the polh promoter in

permissive and non-permissive cell lines.