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Baculoviruses

Introduction

The virus familyBaculoviridae have been known for hundreds of years. The earliestwritten accounts of baculovirus infections describe the infection of Chinese silkworms.

The key features of many descriptions include the paralysis and subsequent liquefaction

of the afflicted larvae. The liquefaction allows for the release of progeny virus producedfrom the insect biomass.

It wasn't until the early 20th century that it was established that the virus particles were

embedded in proteinaceous crystals of polyhedrin. This crystalline matrix allows the

virus to survive in the environment. It was at this stage that baculoviruses were suggestedas a method of natural control of pest insect populations.

In the 1930's and 1940's rod shaped virions were identified within the crystallinepolyhedrin. During the same period baculoviruses were observed to be an effectivebiological control agent of an insect pest. It was discovered that the spruce sawfly

(accidentally introduced into North America) could be effectively controlled by the

subsequent introduction of a baculovirus.

The first baculovirus to be registered as a pesticide (in 1975) was a commercial failure.However the use of a baculovirus as a pest control agent was a nucleopolyhedrovirus

used to control the Douglas fir tussock moth in 1984 was a notable success. This lead to

increased efforts to understand the molecular biology of the baculovirus which in turn

lead to renewed industrial interest in the development of baculovirus pesticides in the

1990's.

Major advancements have been made in the field of baculovirology in the past two

decades. These viruses now have a major role in the field of biomedical research as wellas contributing to our understanding of the complex virus-host interactions.

Baculoviruses are now used for expression of heterologous genes and the development of

cell culture techniques has made large scale production of baculovirus for insect control

possible.

Classification

Members of theBaculoviridae are divided into two genera, Granulovirus andNucleopolyhedrovirus. The generaNucleopolyhedrovirus can be further divided into two

groups morphologically, based on the number of nucleocapsids per virion. The single-nucleopolyhedroviruses (SNPV) contain a single nucleocapsid per virion, whereas the

multiple-nucleoplyhedroviruses (MNPV) contain multiple nucleocapsids per virion. Both

the SNPVs and MNPVs can contain numerous virions per inclusion body.


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The general classification of theBaculoviridae is outlined below.

Classification of baculoviruses:

Family: Baculoviridae

Genus: Granulovirus (GV)

Nucleopolyhedrovirus (NPV)

Groups within the NPVgenus:

Multiple-embedded nucleopolyhedroviruse.g.Autographa californica MNPV

(AcMNPV)

Single-embedded nucleopolyhedrovirus e.g.Trichoplusia ni SNPV (TnSNPV)

The members of the Granulovirus genus (GVs) can be distinguished from the members

of theNucleopolyhedrovirus genus (NPVs) morphologically. The GVs have a granularappearance, whereas the NPVs have a more crystalline structure. GVs only contain a

single virion per inclusion body.

The majority of the information below will focus on NPVs as more information available

on this genus.

Genome

Baculoviruses have a circular, double stranded DNA genome. The genome size of these

viruses range in size from 80 - 180 kbp. Of the fully sequenced baculovirus genomes the

number of open reading frames (orfs) ranges from approximately 120 to 160. In additionto the genes encoded in the genome the are also a number of small repeated sequences

known as homologous regions (hrs) interspersed in the genome. These regions have been

shown to enhance early gene transcription and also to act as origins of replication. Manyof the genes in a baculovirus genome have overlapping ends allowing a large number of

genes to be encoded in a smaller amount of DNA. A diagram of theEppo MNPV genome

map is shown below.


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Structure

Baculoviruses have a distinctive rod shaped nucleocapsids that are 30-60nm in diameterand 250-300nm in length. GVs are occluded in ovicylindrical bodies with dimensions of

about 0.3m x 0.5m. The occluded form of NPVs are polyhedral in shaped and are

approximately 0.15-15m in size. The occluded form of both the GVs and the NPVs canclearly be seen using a light microscope.

NPV polyhedra (left) and a cross section of an MNPV

The occlusion derived virus (ODV) is the form of the virus which is produced in the latter

stages of viral infection and is enclosed in a proteinaceous occlusion body. They allow

for horizontal spread of the virus from insect to insect and allow the virus to persist for

long periods in the environment.


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Baculoviruses also have a second morphology. This second form of the virus is found

within an infected insect and in tissue culture. This form is know as budded virus (BV).

BVs generally contain a single nucleocapsid and are enclosed in an envelope obtained asthe nucleocapsids bud out through the cell wall. Prior to the budding of the virus the cell

wall is modified by the addition of the viral protein GP64. This protein has been shown to

be required for effective spread of the virus within the host.

Replication

Baculoviruses have a biphasic replication cycle. As described above there is onemorphological form of the virus that is important for the horizontal spread of the virus in

the environment (occluded virus, ODV) and a second form which is involved in the

spread of the viral infection within the host (budded virus, BV).

The general process of a baculoviral infection begins with ingestion of the occlusionbodies (OB) on the diet. Once the OB reaches the midgut of the insect the alkaline pH

causes the dissolution of the occlusion body releasing the virions (ODV). The released

virions then pass through the peritrophic membrane of the the midgut. It has beensuggested that there may be some baculoviral proteins incorporated into the ODV which

may enhance the ability of the virions to pass through the peritrophic membrane. These

proteins include forms of chitinases and metalloproteases. The basic virus infectionprocess is shown in the following diagram.
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For an animation of the NPV infection cycle, click on the caterpillar below (requiresFlash plugin)

Once the virions have entered the midgut epithelia the nucleocapsids migrate to the

nucleus of the cell. This migration may be in association with the cellular actin. Once thenucleocapsids reach the nucleus the DNA is uncoated into the nucleus. Phosphorylation

of the DNA binding protein p6.9 causes the DNA to unwind allowing expression and

replication of the viral genome. This process is slightly different for the granuloviruses.These viruses release their DNA into the nucleus through the nuclear pore. For MNPVs it

is postulated that some of the nucleocapsids may bypass the nucleus and bud out of the

cell, allowing the virus to infect other cells faster than if they have to replicate the

genome. The following table out line the various phases ofbaculovirus replication. Formore information on each stage click here or on the name of the phase.

Phase: Description:

Immediate early Expression of viral transregulators and genes which do not

require transregulators for efficient transcription. Many of

the genes expressed in this phase are involve in establishing

the infection.

Delayed early Expression of genes involved in the replication of the virus

and manipulation of the host. Delayed early genes often

require the presence of viral transregulators (e.g. IE-0, IE-1,PE38) for efficient transcription.

Late Transition from early to late is characterised by shutdown of
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the host cell DNA replication and protein synthesis.

Nucleocapsids are produced. Budded virus is produced and

disseminates the virus throughout the host.

Very late (or occlusion) Advanced stage of virus infection. Virions become occludedin the protein polyhedrin. Viral proteases liquefy the host

and degrade the chitinous exoskeleton. Occluded progeny

virus is disseminated onto surrounding material for

horizontal spread.

An general overview of the replication cycle of baculoviruses is shown below.

Proteins

The following table shows a selection of proteins that have been shown to have animportant role in a baculovirus infection. A brief description of the function of the protein

is also shown.

Protein: Function:

Polyhedrin/Granulin Hyper-expressed protein which produces the crystalline matrixof the occlusion bodies. Provides protection from environmentaldamage

GP64/F-protein Present on budded virus only; Envelope fusion protein required

for efficient entry of the budded virus into cells.

EGT Enzyme for inactivating the host moulting hormones,

ecdysteroids.
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P35, IAP-1, -2, -3,

-4

Inhibitors of apoptosis - prevent or delay cells from undergoing

programmed cell death.

DNApol Viral DNA polymerase - Required to replicate the viral genome.

IE-0, -1, -2, PE38 Transactivators produced early in the replication cycle. Regulate

the activity of other genes especially early in the replicationcycle.

LEFs (at least 18) Late expression factors - required for the expression of late

genes. Some also act to down-regulate host cell activities

P6.9 Dephosphorylation of this protein is required for DNA

packaging. Phosphorylation on viral entry into the cell leads tothe DNA unwinding.

Ubiquitin Has similarity to eukaryotic ubiquitin. May act by blocking the

degradation of selected proteins during viral infection

Cathepsin and

Chitinase

Possible role in damaging peritrophic membrane to aid initial

infection. Required for liquefaction of the host and hencedissemination of the progeny virus

Translation

Most of the very early and early proteins are transcribed and translated as a single

protein. Once the replication of the baculovirus moves into the late phase it is more

common to have di- or polycistronic transcripts. With these transcripts it is often only the

first protein in the transcript that is translated, although the other protein(s) are sometimestranslated, although this is usually with much lower efficiency.

A number of proteins in baculoviruses require post-translational modifications to functioncorrectly. These modifications include glycosylation, phosphorylation, cleavage of leader

sequence and formation of multimers.

Virus/Host Interactions

Baculoviruses interact with their insect host in a number of ways. The initial, most

obvious interaction is the dissolution of the protective proteinaceous matrix in the midgutof the host larvae. There are a number of other, more complex interactions. The best

studied baculovirus/host interactions include: apoptosis and apoptosis inhibition;

prevention of host moulting by EGT activity and the action of viral chitinases.

The purpose of the manipulation of the host biology is to increase the the chance of

establishing a viral infection and to all for the production of the maximum amount of

progeny virus - i.e. maximum virus production from the insect biomass. The following
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table has some examples of baculoviral proteins which are involved in the manipulation

of the host biology and their function. For further information on any of these proteins

eitherclick hereor on the protein name below.

Protein: Function:

EGT Inactivates the host moulting hormones by conjugating aUDP-sugar group to them. Reduces stress on the insect and

prevents sloughing/apoptosis of midgut cells.

P35; IAP-1, -2, -3, -4 Apoptosis inhibitors. Prevent the process of "programmed

cell death" which is a response to viral infection (among otherthings)

Chitinase/Cathepsin Involved in the liquefaction of the host and hence the

dissemination of the occluded form of the virus for horizontal

spread.

Tropism

Members of theBaculoviridae family have various tissue tropisms. The NPVs which

affect lepidopteran insects (caterpillars) tend to infect all of the major tissue types. They

initially infect the midgut cells before budding out and infecting other tissues such as

haemocytes, fat bodies, the epidermis and the tracheal matrix. Very few if any occlusionbodies are formed in the midgut cells. Most of the ODV is produced in the other tissues.

Almost all of the other baculoviruses only infect midgut cells (and produce occludedvirus in these cells). A small number of the GVs (including the type species, the Cydia

pomenella GV) which have a cellular tropism similar to the lepidopteran NPVs, i.e.

initially infect the midgut epithelium and then spread to almost all other tissues and

produce occluded virus in those tissues.

Uses

Baculoviruses have been used for decades as a biological pesticide to control agricultural

pests. The research into the biology of these viruses and ways of improving them as a

pest control method has lead to the use of these viruses as a heterologous protein

expression system. Large amounts of protein with all the normal eukaryotic post-translational modifications can be produced.
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