Microbiology Bytes

Baculoviruses Introduction The virus family Baculoviridae have been known for hundreds of years. The earliest written 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 produced from 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 suggested as a method of natural control of pest insect populations. In the 1930's and 1940's rod shaped virions were identified within the crystalline polyhedrin. During the same period baculoviruses were observed to be an effective biological 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 well as 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 the Baculoviridae are divided into two genera, Granulovirus and Nucleopolyhedrovirus. The genera Nucleopolyhedrovirus can be further divided into two groups morphologically, based on the number of nucleocapsids per virion. The singlenucleopolyhedroviruses (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.

The general classification of the Baculoviridae is outlined below. Classification of baculoviruses: Family: Genus: Groups within the NPV genus:

Baculoviridae Granulovirus (GV) Nucleopolyhedrovirus (NPV) • Multiple-embedded nucleopolyhedrovirus e.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 the Nucleopolyhedrovirus genus (NPVs) morphologically. The GVs have a granular appearance, 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 addition to 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. Many of 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 the Eppo MNPV genome map is shown below.

Structure Baculoviruses have a distinctive rod shaped nucleocapsids that are 30-60nm in diameter and 250-300nm in length. GVs are occluded in ovicylindrical bodies with dimensions of about 0.3µm x 0.5µm. The occluded form of NPVs are polyhedral in shaped and are approximately 0.15-15µm in size. The occluded form of both the GVs and the NPVs can clearly 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.

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 as the 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 one morphological 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 occlusion bodies (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 been suggested 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 infection process is shown in the following diagram.

For an animation of the NPV infection cycle, click on the caterpillar below (requires Flash 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 the nucleocapsids 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 of baculovirus replication. For more 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

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 occluded in 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 an important 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 matrix of the occlusion bodies. Provides protection from environmental damage 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.

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 replication cycle.

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 to the 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 hence dissemination 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 sometimes translated, although this is usually with much lower efficiency. A number of proteins in baculoviruses require post-translational modifications to function correctly. 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 midgut of 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

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 either click here or on the protein name below. Protein:

Function:

EGT

Inactivates the host moulting hormones by conjugating a UDP-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 other things)

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 the Baculoviridae 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 occlusion bodies 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 occluded virus 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 posttranslational modifications can be produced.