Virus in Mol Bio

• Viruses in molecular biology include:
  • HIV
  • Hepatitis B
  • Ebola Virus
  • Adenovirus
  • Influenza
  • Bacteriophage

Baltimore System of Classification of Viruses

• Developed by David Baltimore.
• A virus classification system that groups viruses into families based on:
  • Type of genome (DNA, RNA).
  • Single-stranded (ss) or double-stranded (ds).
  • Method of replication.

Viral Morphology

• Helical morphology:
  • Seen in nucleocapsids of many filamentous and pleomorphic viruses.
  • Consist of a helical array of capsid proteins (protomers) wrapped around a helical filament of nucleic acid.
• Spherical viruses:
  • Have an icosahedron structure: a polyhedron having 20 equilateral triangular faces and 12 vertices.
  • Protomers (structural polypeptide chains) are arranged in oligomeric clusters called capsomeres.
  • Arrangement permits classification by capsomere number and pattern.
• Lambda phage particle:
  • Consists of a head and a tail that can have tail fibers.
  • Composed of 12–14 different proteins with more than 1000 protein molecules total.
  • Contains one DNA molecule located in the phage head.

RNA Viruses

• RNA viruses comprise 70% of all viruses, varying remarkably in genome structure.
• High mutation rates:
  • Due to the error rate of enzymes involved in RNA replication.
  • Mutation rates of $10^{-4}$ lead to continuous generation of virus variants, showing great adaptability to new hosts.
• Genome:
  • May be single-stranded (ss) or double-stranded (ds).
  • May occupy a single RNA segment or be distributed on two or more separate segments (segmented genomes).
• Classification according to RNA sense or polarity:
  • Negative-sense
  • Positive-sense
  • ambisense RNA viruses.
• Positive-sense viral RNA:
  • Similar to mRNA.
  • Can be immediately translated by the host cell.
• Negative-sense viral RNA:
  • Complementary to mRNA.
  • Must be converted to positive-sense RNA by an RNA-dependent RNA polymerase before translation.

Double-Stranded RNA Viruses

• Diverse group varying widely in:
  • Host range (humans, animals, plants, fungi, and bacteria).
  • Genome segment number (one to twelve).
  • Virion organization (Triangulation number, capsid layers, spikes, turrets, etc.).
• Examples:
  • Rotaviruses: cause gastroenteritis.
  • Picobirnaviruses.
  • Bluetongue virus: an economically important pathogen of cattle and sheep.
• Reovirus family:
  • Contain 10, 11, or 12 separate genome segments.
  • Code for 3 enzymes involved in RNA replication, 3 major capsid proteins, and smaller structural proteins.

DNA Viruses

• Some DNA viruses contain a single genome of linear dsDNA.
• Papovaviruses:
  • Include polyoma- and papillomaviruses.
  • Have circular DNA genomes, about 5.1 and 7.8 kb pairs in size.
  • DsDNA serves as a template both for mRNA and for self-transcription.
• Parvovirus family:
  • Single-stranded linear DNA, 4–6 kb in size.
• Adeno-associated virus (AAV):
  • Incapable of producing progeny virions except in the presence of helper viruses (adenovirus or herpesvirus).
  • Replication defective.
• Circovirus family:
  • Circular single-stranded DNA of only 1.7 to 2.3 kb.
  • Smallest autonomously propagated viruses.
  • Isometric capsid measures 17 nm and is composed of 2 protein species only.

Plant Viruses

• Over 50% of known plant viruses are rod-shaped (flexuous or rigid).
  • The length of the particle is normally dependent on the genome but it is usually between 300–500 nm with a diameter of 15–20 nm.
• Protein subunits can be placed around the circumference of a circle to form a disc.
  • In the presence of the viral genome, the discs are stacked, then a tube is created with room for the nucleic acid genome in the middle.
• The second most common structure amongst plant viruses are isometric particles.
  • They are 25–50 nm in diameter. In cases when there is only a single coat protein, the basic structure consists of 60 T subunits, where T is an integer.
• Some viruses may have 2 coat proteins that associate to form an icosahedral shaped particle.
• There are three genera of Geminiviridae that consist of particles that are like two isometric particles stuck together.
• A very small number of plant viruses have, in addition to their coat proteins, a lipid envelope.

Animal Viruses

• Viruses of vertebrates are informally distinguished between those that primarily cause infections of humans and those that infect other animals.
• Different viruses can infect all the organs and tissues of the body, with outcomes ranging from mild or no symptoms to life-threatening diseases.
• Baculoviridae:
  • A family of viruses that infect arthropods, lepidoptera, hymenoptera, diptera, and decapoda serve as natural hosts.
  • Baculoviruses are incapable of replicating within the cells of mammals and plants.

Viral Envelopes

• Some viruses have viral envelopes covering their protective protein capsids.
• Envelopes are typically derived from host cell membranes (phospholipids and proteins) but include some viral glycoproteins.
• They may help viruses avoid the host immune system.
• The cell from which the virus buds will often die or be weakened and shed more viral particles for an extended period.
• Lipid bilayer envelope:
  • Relatively sensitive to desiccation, heat, and detergents.
  • Easier to sterilize than non-enveloped viruses, have limited survival outside host environments, and typically must transfer directly from host to host.
• Enveloped viruses:
  • Possess great adaptability and can change quickly to evade the immune system.
  • Can cause persistent infections.

Life Cycle of a Bacteriophage

• Attachment to host cell and injection of lambda DNA.
• Lambda DNA circularizes.
• Lytic pathway:
  • Rapid replication of lambda DNA and its packaging into complete viruses.
  • Synthesis of viral proteins needed for the formation of new viruses.
  • Cell lysis releases a large number of new viruses.
• Prophage pathway:
  • Integration of lambda DNA into host chromosome.
  • Integrated lambda DNA replicates along with host chromosome.
  • Cell division.
  • Induction event.

Bacterial Viruses in Biochemical and Genetic Research

• DNA Phages of the T Series:
  • T phages of E. coli are large lytic phages that contain a single molecule of double- stranded DNA.
  • This molecule is about $2 × 10^5$ base pairs long in T2, T4, and T6 viruses and about $4 × 10^4$ base pairs long in T1, T3, T5, and T7 viruses.
  • T-phage virions consist of a helical protein “tail” attached to an icosahedral “head” filled with the viral DNA.
• Bacteriophage λ:
  • It infects E. coli and typifies the temperate phages.
  • This phage has one of the most studied genomes and is used extensively in DNA cloning.
  • On entering an E. coli cell, the double-stranded λ DNA assumes a circular form, which can enter either the lytic cycle (as T phages do) or the lysogenic cycle.
• Small DNA Phages:
  • The genome of some bacteriophages encodes only 10 – 12 proteins.
  • These small DNA phages are typified by the ΦΧ174 and the filamentous M13 phages.
  • These were the first organisms in which the entire DNA sequence of a genome was determined, permitting extensive understanding of the viral life cycle.
• RNA Phages:
  • Some E. coli bacteriophages contain a genome composed of RNA instead of DNA.
  • These viruses, among the smallest known, encode only four proteins: an RNA polymerase for replication of the viral RNA, two capsid proteins, and an enzyme that dissolves the bacterial cell wall.

Bacteriophage λ

• From 1970, bacteriophage λ was used as a cloning vector.
  • More than 400 variants of lambda phage are developed.
• The genome of lambda phage contains a ds DNA molecule of 48,502 bp.
• The DNA molecule contains two single-stranded termini of 12 nucleotides (cohesive termini or cos).
• After entering the host, the cohesive termini associate with the other end and with the help of host ligase and gyrase form a closed circular DNA molecule.
• During the lytic cycle, the circular DNA directs the synthesis of approximately 30 proteins to execute DNA replication, assembly of bacteriophage, and host cell lysis.
• In the lysogenic cycle:
  • λ DNA is integrated into the host chromosome, and very few genes get transcribed.
  • Important genes include:
  • cI: inhibits lytic function and regulates λ phage gene transcription.
  • int: integrates phage DNA to host chromosome.
  • rex A and rex B: prevent infection by other bacteriophages.

Filamentous Phages

• Filamentous phages are not lytic; they coexist with the infected cells for several generations.
• Among the filamentous phages, fd, fl, and M13 have been well characterized, and their genomes have been sequenced.
• M13:
  • Filamentous bacteriophage and composed of circular single-stranded DNA (ssDNA) (6407 nucleotides).
  • Encapsulated in approximately 2700 copies of the major coat protein P8 and capped with 5 copies of two different minor coat proteins (P9, P6, P3) on the ends.
  • The minor coat protein P3 attaches to the receptor at the tip of the F pilus of the host E. coli.
• Infection with filamentous phages is not lethal, as it is a non-lytic virus.

Insertion into Host - Bacteriophage Lambda

• Bacteriophage Lambda binds to an E. coli cell through its J protein in the tail tip.
• The J protein interacts with the maltose outer membrane porin (the product of the lamB gene) of E. coli.
• The linear phage genome is injected through the outer membrane.
• The DNA passes through the mannose permease complex in the inner membrane and immediately circularizes using the cos sites, 12-base G-C-rich cohesive "sticky ends".
• The single-strand viral DNA ends are ligated by host DNA ligase.

Bacteriophage Lambda - Transcription and Lifecycle

• Transcription starts from the constitutive PL, PR, and PR' promoters, producing the 'immediate early' transcripts.
• At first, these express the N and cro genes, producing N, Cro, and a short inactive protein.
• Cro binds to OR, preventing access to the PRM promoter, preventing expression of the cI gene.
• The N protein is an antiterminator and functions to extend the reading frames to which it is bound.
• The extended transcripts (the 'late early' transcripts) include the N and cro genes along with cII and cIII genes.
• The cIII protein acts to protect the cII protein from proteolysis by FtsH.

Bacteriophage Lambda - Lifecycle Determination

• On initial infection, the stability of cII determines the lifestyle of the phage.
  • Stable cII will lead to the lysogenic pathway.
  • If cII is degraded, the phage will go into the lytic pathway.
• The 'late early' transcripts continue being written, including xis, int, Q, and genes for replication of the lambda genome (OP).
• Cro dominates the repressor site, repressing synthesis from the PRM promoter (which is a promoter of the lysogenic cycle).
• Rightward transcription expresses the O, P, and Q genes.
  • O and P are responsible for initiating replication.
  • Q is another antiterminator that allows the expression of head, tail, and lysis genes from PR.

Bacteriophage Lambda - Lysogenic Lifecycle

• The lysogenic lifecycle begins once the cII protein reaches a high enough concentration to activate its promoters after a small number of infections.
• The 'late early' transcripts continue being written, including xis, int, Q, and genes for replication of the lambda genome.

Prophage Integration

• The integration of phage λ takes place at a special attachment site in the bacterial and phage genomes, called attλ.
• The sequence of the bacterial att site is called attB, between the gal and bio operons, and consists of the parts B-O-B', whereas the complementary sequence in the circular phage genome is called attP and consists of the parts P-O-P'.
• The integration itself is a sequential exchange via a Holliday junction and requires both the phage protein Int and the bacterial protein IHF (integration host factor).
• Both Int and IHF bind to attP and form a DNA-protein-complex designed for site-specific recombination of the phage and host DNA.
• The original B-O-B' sequence is changed by the integration to B-O-P'-phage DNA-P-O-B'.
• The phage DNA is now part of the host's genome.

Lysogeny Maintenance

• Lysogeny is maintained solely by cI.
  • cI represses transcription from PL and PR while upregulating and controlling its own expression from PRM.
  • It is, therefore, the only protein expressed by lysogenic phage.
• The prophage is duplicated with every subsequent cell division of the host.
• The phage genes expressed in this dormant state code for proteins that repress the expression of other phage genes (such as the structural and lysis genes) in order to prevent entry into the lytic cycle.
• These repressive proteins are broken down when the host cell is under stress, resulting in the expression of the repressed phage genes.
• Stress can be from starvation, poisons (like antibiotics), or other factors that can damage or destroy the host.
• In response to stress, the activated prophage is excised from the DNA of the host cell by one of the newly expressed gene products and enters its lytic pathway.

Bacteriophage as a Vector - Reasons for Use

• Acceptance of large foreign DNA fragments by the phage increases the chances of screening a single clone carrying a DNA sequence corresponding to a complete gene.
• Development and availability of refined techniques aimed at minimizing the problems of background due to non-recombinants.
• The possibility of screening several thousand clones at a time from a single petri plate.
• The ease with which the phage library can be stored as a clear lysate at 4°C for months without significant loss in plaque-forming activity.

Bacteriophage as a Vector - Problems with Use

• The sequence of wild-type strains contains multiple restriction enzyme sites.
• Those sites are often located in regions essential for lytic growth.
• The phages cannot accommodate DNA molecules longer than the viral genome.

Types of Virus-Based Vectors & Application

• Insertion vectors:
  • Vectors with a single site for inserting foreign DNA.
  • The genome of these bacteriophages is 20% shorter than its wild-type ancestor because many genes have been removed.
  • The maximum size of DNA that can be accommodated within an insertion vector varies from 5-11 kb.
• Replacement vectors:
  • Bacteriophage having a pair of cloning sites flanking a segment of nonessential DNA are known as replacement vectors.
  • The largest segment of foreign DNA that can be cloned varies from 8-24 kb.
• Expression vectors:
  • From the early 80’s, several bacteriophage expression vectors have been developed for expression in bacterial systems.
  • For example, the ZAP express system (Stratagene) can be used to clone 12 kb DNA, which can be further expressed on the bacterial system (via lac promoter) and in the mammalian system (cytomegalovirus promoter).
• Application:
  • Basic research
  • Gene therapy
  • Vaccine production

Viral Vector Systems and their properties

• Adenovirus (~36 kb genome)
  • E1 deleted, replaced by the expression cassette.
  • 7-8 kb max capacity.
• Adeno-associated virus (4.7 kb genome)
  • Expression cassette inserted, deleted of its current contents.
  • 4.5 kb max capacity.
• Retrovirus (7-10 kb genome)
  • Expression cassette inserted, Self-inactivating 3' LTR.
  • 8 kb max capacity.
• Lentivirus (9-10 kb genome)
  • Expression cassette inserted, Self-inactivating 3' LTR, includes CPPT and CTS.
  • 8 kb max capacity.
• Liposome + plasmid (unlimited sized genome)
  • Expression cassette inserted, includes ori and antibiotic resistance gene
• Unlimited genome size capacity

Gene Therapy Workflow and Viral Vectors

• Cells harvested from the patient.
• In lab, the virus is altered so it cannot reproduce.
• A gene is inserted into the virus.
• Altered virus mixed with patient's cells.
• Cells become genetically altered.
• Altered cells injected into the patient's body.
• Altered cells produce the desired protein.

Key properties of Viral Vectors

Cell State, Viral System, and Expression Types.

Plating Bacteriophage λ

Requirements

1. Plating materials (nutrient media, agar, agarose ).
2. Bacteriophage of interest.
3. E. coli strain.

Growing bacteria

1. Inoculate nutrient broth (LB) with a single colony of E. coli strain.
2. Grow the culture overnight at 37° C with agitation.
3. Collect the bacteria by centrifuging the culture at 5000-6000 rpm for 10 min.
4. Discard the supernatant and re-suspend the pellet in phosphate buffered saline or MgSO4 solution (sterile).
5. Store the bacteria at 4°C.

Plating

1. Dilute the bacteriophage solution (10 fold) into suspension medium with gelatin (SMGel).
2. Aliquot 100 µl of bacteriophage into a sterile tube and mix with stored bacteria by vortexing.
3. Incubate the mixture at 37° C for 20 min.
4. Add molten agarose (~40°C) to the tube and mix well by gentle tapping.
5. Pour the mixture on an agar plate and swirl the plate for even distribution.
6. Allow the agarose (top agar) to harden at room temperature.
7. Incubate the plate at 37° C overnight.
   -Usable bacterial strains are HB101, NM522, X1-1 Blue, SMR10, Tap90, NM531 etc.

Picking Bacteriophage λ Plaques

Requirements

• Bacteriophage grown (plaques) plate.
• Suspension medium (MgSO4, Tryptone or Casein, Sodium chloride, yeast extract).

Process

1. Aliquot 1 ml SM in a microcentrifuge tube with a drop (~50 µl) of chloroform.
2. Identify a well-separated plaque and collect the plaque along with the agar below by sucking through a pipette tip (Isolation can be done using a sterile toothpick/stick/applicator also).
3. Add the collected agar/agarose slab to the SM/chloroform mixture immediately.
4. Incubate the tube at room temperature for 1-2 hr to allow the bacteriophage to diffuse.

Large-Scale Growth of Bacteriophage λ

Requirements

• Stock of Bacteriophage.
• Bacterial strain.
• Nutrient medium (LB, NZCYM).

Process

1. Grow bacterial strain in 100 ml nutrient broth overnight at 37° C.
2. Make 4 aliquots (25 ml) and centrifuge the solution.
3. Re-suspend the pellet in 3-4 ml of SM.
4. Add bacteriophage particles and incubate for 20 min at 37° C.
5. Add the mixture to 500 ml of nutrient medium and incubate at 37° C with vigorous shaking for 8-12 hr.
6. Monitor the culture for complete lysis (presence of large bacterial debris).

Isolation of Bacteriophage λ Particles by PEG

Requirements

1. Bacteriophage-infected E. coli culture.
2. Chloroform, NaCl, Polyethele glycol (PEG 8000), suspension medium.
3. Pancreatic DNase, Pancreatic RNAse.

Process

1. Add pancreatic DNase and RNase (1µg/ml) to the lysed culture and incubate for 30 min at room temperature (RT).
2. To each 500 ml culture, add 1M NaCl and dissolve the salt.
3. Centrifuge the culture at 9000-10000 rpm for 10 min at 4°C.
4. To the clear supernatant, add solid PEG 8000 (10% w/v), dissolve the PEG by stirring at RT.
5. Transfer the solution to centrifuge bottles and keep on ice for 1 hr to facilitate precipitation of bacteriophage particles.
6. Centrifuge the solution at 9000 rpm for 10 min at 4°C and collect the precipitation.
7. Re-suspend the pellet in suspension medium (8ml for 500 ml culture) for 1 hr at RT.
8. Add equal volume (8 ml) of chloroform and vortex the mixture.
9. Centrifuge the mixture to separate organic and aqueous phase at 4500-5000 rpm for 15 min at 4°C.
10. Collect the aqueous phase, which contains the bacteriophage particles.

Isolation of Bacteriophage λ Particles by CsCl Gradients

• CsCl or glycerol-mediated isopycnic centrifugation processes are used to isolate the highly purified bacteriophage particles.

Requirements

• CsCl solid to prepare different density solution in SM.

Process

1. Prepare three CsCl gradients (1.45g/ml, 1.50g/ml, 1.70 g/ml).
2. Add 0.5 gm of solid CsCl to 1 ml of bacteriophage suspension and dissolve the CsCl.
3. Pour the gradient solution (decreasing density on top).
4. Centrifuge the tubes at 22000 rpm for 2 hr at 4°C and collect the pure bacteriophage from the 1.50 g/ml layer.

Isolation of Bacteriophage λ Particles by Glycerol Gradients

Requirements:

• Suspension medium, Glycerol, EDTA, Pancreatic DNase, Pancreatic RNase, Suspension of bacteriophage.

Process:

1. Prepare glycerol gradient step with 40% (bottom) and 5% (middle) glycerol in a tube.
2. Lay the bacteriophage solution on top of the 5% layer and fill the tube with SM.
3. Centrifuge the tube at 35000 rpm for 1 hr at 4°C.
4. Discard the supernatant and resuspend the pellet in 1 ml of SM.
5. Add pancreatic DNase and RNase to the solution and incubate at 37°C for 30 min.
6. Add EDTA to the solution (20 mM) to deactivate the enzymes.

Isolation of Bacteriophage λ Particles by Centrifugation

Requirements:

• Suspension medium, bacteriophage suspension.

Process:

1. Transfer the bacteriophage suspension into a tube and collect the particles by centrifugation at 25000 rpm for 2 hr at 4°C.
2. Discard the supernatant and add 1-2 ml of SM to the pellet.
3. Store it overnight at 4°C on a rocking platform.
4. Next day, pipette the solution up and down to ensure the re-suspension of the particles.

Extraction of Bacteriophage λ DNA

• DNA isolation can be done by degrading the coat protein and separating the DNA from the protein particles.

Proteinase K and SDS method

Requirements:

• Chloroform, phenol, Dialysis buffer (DB), EDTA, Ethanol, SDS, Sodium accetate, Proteinase K, Bacteriophage particles.

Process:

1. Transfer the bacteriophage suspension to a dialysis tube and put it into DB at RT for 1 hr.
2. Add 0.5 M EDTA (final concentration 20 mM) to the dialyzed solution.
3. Add proteinase K (50 µg/ml) and SDS (0.5%) and mix the solution gently.
4. Incubate the bluish solution for 1 hr at 56 °C and cool it to RT.
5. Add equal volume of phenol (tris equilibrated) and mix them gently to form an emulsion.
6. Centrifuge the tube at 5000 rpm for 5 min at RT to separate the aqueous and organic phase.
7. Collect the aqueous phase and extract with a 1:1 mixture of equilibrated phenol: chloroform.
8. Recover the aqueous phase and add ice-cold ethanol to precipitate the DNA.
9. Re-suspend the DNA in TE buffer (small scale).
10. For CsCl purified DNA, the sample from step 8 will be dialyzed against TE buffer overnight at 4°C.

Extraction of Bacteriophage λ DNA - Formamide Method

Requirements:

• Formamide, EDTA, Ethanol, NaCl, TE buffer, Tris-Cl, Bacteriophage particles

Process:

1. Add 0.1 volume of 2M Tris, 0.05 volume of 0.5M EDTA, and 1 volume of deionized formamide to the bacteriophage particle, and incubate at 37°C for 30 min.
2. Precipitate the DNA by adding 1 volume of water and 6 volumes of ethanol.
3. Collect the pellet with 70% ethanol and centrifuge briefly (10 s).
4. Discard the supernatant and dissolve the pellet in 5M NaCl (6 µl) and ethanol (750 µl).
5. Collect the precipitated DNA and store it in TE buffer.

Rapid Purification of λ DNA from Plate Lysates

Requirements:

• Chloroform, Ethanol, High salt buffer (Tris-HCl, NaCl, EDTA), Isopropanol, Low salt buffer (Tris-HCl, NaCl, EDTA), Phenol: chloroform, SM, TE, resin beads, column. Bacteriophage-infected E. Coli agar plate.

Process:

1. Isolate different numbers (8-10) of well-separated plaques from the plate and put them in 1 ml of SM and 50 µl of chloroform, and incubate at 4°C for 4-6 hr to allow the bacteriophage particles to diffuse out.
2. Add 50-100 µl of this suspension to 150 µl of plating bacteria, incubate that mixture at 37°C for 20 min, and mix with molten agarose to prepare top agar plates.
3. Incubate the plates at 37°C to form enough plaques to cover the plate.
4. Add 7 ml of TM to the plate surface to elute the bacteriophage particles for 4-5 hr of incubation at 4°C.
5. Transfer the eluted solution and centrifuge at 5000 rpm for 10 min at 4°C, and add resin beads to the clear solution for adhesion of bacteriophage, and Centrifuge the bead slurry for 5000 rpm, and transfer the supernatant to a phenol:chloroform mixture.
6. Centrifuge and transfer the aqueous phase with an equal volume of isopropanol, and store at -70°C for 10 min, and Centrifuge it at 12000 rpm for 20 min at 4°C.
7. Collect the pellet and air dry it, and dissolve it in 2 ml of low salt buffer.
8. Further cleanup can be done by column purification.

Long Term Storage of Phage

• Different preservation methods, such as the storage of crude or purified phage lysates at 4 °C, freezing and storage at −80 °C or in liquid nitrogen, and finally storage of dried or lyophilized phages, have been used in different laboratories.
• Unfortunately, none of these methods appeared universal due to the differences in the sensitivity of individual phages to physical conditions and the content of storage media.
• Only ten out of nineteen phages from nine families tested survived over 10 years without a loss of phage titer, but the most appropriate method of storage was not the same for all these phages.

Baculovirus

• Baculoviruses are the most prominent viruses known to affect the insect population.
• They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid.

Baculovirus Replication

• Wild-type Baculovirues exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication.

1. Early Phase:

• In this phase, the virus prepares the infected cell for viral DNA replication.
• Actual initial viral synthesis occurs 0.5 to 6 h after infection.

2. Late Phase:

• In this phase, late genes that code for replication of viral DNA and assembly of virus are expressed.
• Between 6 and 12 h after infection, the cell starts to produce extracellular virus (EV), also called non-occluded virus (NOV) or budded virus (BV).
• Peak release of extracellular virus occurs 18 to 36 h after infection.

3. Very Late Phase:

• In this phase, the polyhedrin and p10 genes are expressed, occluded virus (OV) or occlusion bodies (OB) or polyhedral inclusion bodies (PIBs) are formed, and cell lysis begins.

Recombinant Baculovirus

• The major difference between the naturally occurring in vivo infection and the recombinant in vitro infection is that the naturally occurring polyhedrin gene within the wild-type baculovirus genome is replaced with a recombinant gene or cDNA.
• These genes are commonly under the control of polyhedrin and p10 promoters.

Expression in insect cells:

• BaculoDirect™ Baculovirus Expression System typically requires bacterial transformation.
• Bac-to-Bac® Baculovirus Expression System uses a unique bacmid shuttle vector.
• Bac-to-Bac® HBM Baculovirus Expression System enables secreted protein expression via the honeybee melittin (HBM) secretion signal.
• Bac-N-Blue™ Baculovirus Expression System is the classic and trusted expression system in insect cells.

Generating Recombinant Virus

By Homologous Recombination

• The most common baculovirus used for gene expression is AcMNPV (Autographa californica multiple nuclear polyhedrosis virus).
• AcMNPV has a large (130-kb), circular, double-stranded DNA genome.
• The gene of interest is cloned into a transfer vector containing a baculovirus promoter flanked by baculovirus DNA derived from a nonessential locus, in this case, the polyhedrin gene.
• The gene of interest is inserted into the genome of the parent virus (such as AcMNPV) by homologous recombination after transfection into insect cells.
• Typically, 0.1% to 1% of the resulting progeny are recombinant.
• A higher percentage of recombinant progeny virus (nearly 30% higher) results when the parent virus is linearized at one or more unique sites located near the target site for insertion of the foreign gene into the baculovirus genome.

By Site-Specific Transposition

• A faster approach for generating a recombinant baculovirus uses site-specific transposition with Tn7 to insert foreign genes into bacmid DNA propagated in E. coli.
• The gene of interest is cloned into a pFASTBAC™ vector, and the recombinant plasmid is transformed into DH10BAC™ competent cells, which contain the bacmid with a mini-attTn7 target site and the helper plasmid.
• The mini-Tn7 element on the pFASTBAC plasmid can transpose to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid.

Recombinant Virus Generation by Site-Specific Transposition or column wash

1. Transformation / donor plasmid or bacmid
2. competent BH 10Bac cells
3. Extraction of recombinant bacmid DNA:
4. DNA transfection of insect cells with recombinant bacmid
5. Recombinant virus particles infected into insect cell
6. Extraction of expression varification
7. Virus Stock via Plaque Assays

Purification of Viral DNA techniques

Recombination techniques

-Techniques to purify viral DNA include phenol extraction , cesium chloride purification, or affiinity purification (e.g CONCERT™ High Purity) through the following steps.

CONCERT™ High Purity

1. Apply 2 ml of Equilibration Buffer [600 mM NaCl, 100 mM sodium acetate (pH 5.0), 0.15% Triton X-100] to the column.
2. Pellet 1.5 ml of an overnight culture and add 0.4 ml of Cell Suspension Buffer [50mM Tris-HCl (pH 8.0), 10 mM EDTA, containing RNase A at 0.2 mg/ml] to the pellet and suspend cells until homogeneous.
3. Add 0.4 ml of Cell Lysis Solution [200 mM NaOH, 1% SDS]. Mix gently by inverting the capped tube five times. Do not vortex. Incubate at room temperature for 5 min.
4. Add 0.4 ml of Neutralization Buffer [3.1 M potassium acetate (pH 5.5)] and mix immediately by inverting the tube five times. Do not vortex. Centrifuge the mixture at top speed in a microcentrifuge at room temperature for 10 min. Do not centrifuge at 4°C.
5. Pipet the supernatant from step onto the equilibrated column. Allow the solution in the column to drain by gravity flow. Discard flow-through.
6. Wash the column two times with 2.5 ml of Wash Buffer [800 mM NaCl, 100 mM Sodium acetate (pH 5.0)]. Allow the solution in the column to drain by gravity flow after each wash. Discard flow-through.
7. Elute the DNA by adding 0.9 ml of Elution Buffer [1.25 M NaCl, 100 mM Tris-HCl (pH 8