LECTURE 12: MU, TRANSPOSONS.

ANIMAL VIRUSES: GENERAL FEATURES OF RETROVIRUSES.

4. Mu = a mutator phage

a. Structure:

(i) Virion - icosahedral head, helical tail with tail fibers

(ii) Nucleic acid - dsDNA


The DNA contains ~ 37.2 kb of phage DNA. The ends contain additional DNA taken from the host genome (one end has ~ 50-100 bp; the other end contains ~ 1-2 kb. Host sequences occur in the viral DNA as a result of the packaging mechanism. The virus stuffs DNA into empty phage heads until they are full. The amount of DNA that fits inside Mu phage heads corresponds to slightly more than one viral genome.

b. Some observations:

(i) Mu can vary its host range - the structure of the tail fibers can be altered so that the virus can recognize different hosts

(ii) Mu causes mutations in host DNA

c. Mechanism of host range alteration

The G segment of the Mu genome contains an invertible piece of DNA. If the segment is in the G+ orientation, allowing expression of the genes S and U, then Mu can infect the E. coli strain K12. Arrangement of the segment in the G- orientation permits expression of the genes S' and U', allowing Mu to infect the E. coli strain C.

How common are invertible DNA segments?

This phenomenon appears quite common. Bacteria of the genus Salmonella use invertible DNA segments to regulate host range by changing the type of protein found in their flagella (Figure 7.32). A related mechanism allows yeast to switch its mating type (Figure 7.35).

d. Mechanism of transposition

The genetic material of Mu is inserted into the host genome by transposition - the same process by which transposable elements are inserted into DNA (Figure 6.33):

(i) First, staggered cuts are made in the host DNA by the viral enzyme transposase. The site of insertion is not sequence-specific, so the Mu genome can be inserted anywhere in the host DNA.

(ii) The base pairs within the region where the staggered cuts were made separate thus exposing two single-stranded overhangs.

(iii) The Mu dsDNA is attached to the two single-stranded overhangs. (The ends of Mu containing host DNA are not inserted.)

(iv) The single-stranded regions are "repaired" - gaps are filled in and nicks are ligated. Note that as a consequence of this mechanism, the Mu DNA, now inserted in the host genome, is flanked by "direct repeats" of a short sequence of host DNA.

During lytic growth, the Mu genome is replicated by repeated replicative transpositions with copies of the viral genome generated at various sites in the host genome. The host DNA flanking replicated Mu genomes is cut, releasing viral genomes (with host DNA at the ends) for packaging into particles during assembly.

Essentially, infection by Mu brings a huge "transposon" into the cell. Whereas cellular transposable elements have no independent existence outside of the DNA in which they are inserted, the Mu transposon is capable of an independent existence within the virion particle.

e. Some uses in genetic engineering

(i) The Mu variants known as Mini-Mu are deleted for several genes essential for host cell lysis. They can penetrate cells and insert their genome into the host DNA, but they are unable to produce phage particles or to lyse the cell. These variants are very useful in randomly mutagenizing the host genome when searching for unknown genes.

(ii) The Mini-Mu variant Mud-lac has the lacZ gene added to the viral genome. The lacZ gene may become activated, producing detectable beta-galactosidase, if the viral genome is inserted near an endogenous host promoter. This can be used to identify promoters (and subsequently the genes they regulate) that are active under different physiological conditions.

f. How common is transposition in prokaryotes and eukaryotes?

A wide variety of transposable elements and viruses that use transposition are known. For example,

Transposable elements leave "footprints" behind - direct repeats in the target DNA. This is the hallmark characteristic of insertion events brought about by transposition activity.

Viruses that use transposition to replicate their DNA could be considered "escaped transposons" - transposons that acquired the ability to exist independently as virions by incorporating the sequences required for viral functions within their baggage of genes that they carry around.

I - Eukaryotic viruses

A. An overview of retroviruses

1. Historically important:

a. Retroviruses "overturned" the Central Dogma. It was long thought that DNA transcription to produce RNA was strictly followed by RNA translation to make protein. Retroviruses demonstrated for the first time that it was possible for RNA to be used as a template by an RNA-dependent DNA polymerase to synthesize DNA.

b. Retroviruses were the first viruses discovered to cause cancer. e.g. Rous sarcoma virus, feline leukemia virus

c. Retroviruses are being investigated for use in gene therapy.

d. The retrovirus HIV is responsible for the disease AIDS.

2. Virus particle (Figure 6.48a)

a. Shape: A central core holding the nucleic acid is surrounded by a protein coat and an envelope containing viral proteins.

b. Symmetry: the structure is not especially symmetrical.

3. Genome (Figure 6.48b)

a. Nucleic acid: There are two identical copies of genome, each of which is a (+)strand ssRNA. There are also two t-RNAs that are taken from a previous host cell. The t-RNAs hybridize to the viral RNAs and form bridges that link the four molecules together. Most retroviruses contain ~ 8000 nucleotides; HIV contains 9749.

b. Modifications: the 5' end is capped and the 3' end has a poly-A tail, much like eukaryotic mRNAs.

c. LTRs: The ends of retroviral genomes contain Long Terminal Repeats that are used in replication.

4. Proteins important for retroviruses:

a. Reverse transcriptase - an RNA-dependent DNA polymerase used to make dsDNA from ssRNA.

b. Integrase - inserts the dsDNA intermediate into the host genome; it is thus equivalent in function to the transposase enzyme used by transposable elements and viruses such as Mu.

c. Transcriptional activators (e.g. Tat protein of HIV )

d. Coat proteins (e.g. gp120 glycoprotein of HIV )

5. Path of injection (Figure 6.49)

After penetration of the nucleic acid into the host, dsDNA is produced by reverse transcription in the cytoplasm. The dsDNA then enters the nucleus and is incorporated into the host DNA at random locations, behavior reminiscent of transposons. The viral genome may remain dormant for some time. When the virus plans to lyse the cell, viral transcripts are synthesized and traverse into the cytoplasm where they are translated and packaged during assembly. A lipid bilayer envelope is snatched from the host during the release of viral particles.