Download Lecture Notes: Bacteriophage

Lecture Notes: Bacteriophage
Bacterial viruses contain either a ssRNA, ssDNA, or a dsDNA genome
(rarely a dsRNA genome). Among the ssRNA and ssDNA phage, the
known examples all are positive stranded. The terminology "positive"
and "negative" stranded viral genomes relates to whether the nucleic
acid referred to can form base pair interactions with viral mRNA (which
is defined as the positive strand). A positive strand ssRNA virus has a
genome that acts as mRNA immediately after entering the cytoplasm of
the infected host cell.
The morphology of the viral capsids can be filamentous, icosahedral, or
prolate icosahedral with helical tails (Figs. 10.2, 10.4, 10.5). While
viruses of eukaryotes can be either naked or enveloped (Fig. 10.3),
bacteriophages are only naked (without membrane envelopes).
A typical lytic phage goes through a replication cycle generally
described by Fig. 10.8 of the textbook, starting with the specific
attachment of the virus to a distinct cell surface receptor. There is a
tremendous specificity in the interaction between the virus and the host.
The example described in class of a ssRNA phage is MS2
MS2 is a small, icosahedral phage with a genome size of 3569
nucleotides, with 4 genes: A (maturation) protein, coat protein, replicase
protein, and lysis protein.
Attachment of the phage during infection is at the sides of the F pilus of
E. coli.
As the phage genome is a polycistronic mRNA molecule, the relative
synthesis of the 4 phage proteins during infection is altered by elaborate
translational control, via differential access of ribosome binding sites
(RBS) to the host ribosome. The coat gene has the most readily
accessible RBS on the phage RNA molecule, which acquires complex
secondary structure in the cell. In contrast, the A (maturation) gene can
only be translated during the replication of viral + strands (that is, the
ribosome begins translation of the A gene while the + strand is in the
process of being polymerized; see Fig. 19.2 and Slide 8 from handout).
(I will not ask you about translation of viral lysis protein).
The MS2 replicase protein associates with 3 other host proteins to form
a viral RNA-specific RNA polymerase. There is no DNA intermediate
during MS2 replication. The MS2 replicase is an RNA-templated RNA
polymerase and makes both positive and negative strands of viral RNA.
The only role for viral RNA negative strands is to serve as template for
synthesis of viral positive strands.
The example of a filamentous ssDNA phage is M13.
M13 binds to the tip of the F pilus on E. coli cells containing the F
plasmid.
Unlike other phages described in class, progeny M13 phages are
released from an infected cell without lyzing/killing the host cell. The
phage capsids are assembled as the virus "buds" from the cell, allowing
continued growth of the host (Fig. 19.5).
Since M13 is a positive strand ssDNA phage, there is no synthesis of
viral proteins until after the synthesis of the viral negative strand. The
replication of M13 is very similar to that of the phage X174.
The example of an iscosahedral ssDNA phage is X174.
circular, + ssDNA, 5368 nucleotides in length, encoding 10 genes (Fig.
19.3).
The virus attaches to LPS of the outer cell wall membrane of E. coli and
closely related enteric bacteria.
The viral DNA enters the cell and is converted into a ds circular
molecule by the host DNA synthesis machinery. Host topoisomerases
supercoil the dsDNA, creating the replicative form I (RFI) molecule that
is active for replication of viral + strands.
An early phage protein is CisA, which nicks the + strand of the viral RFI
(at the origin of viral replication) and attaches to the 5' end of the viral
DNA (forming an RFII). This leaves the 3'OH group of the + strand
accessible to prime + strand DNA synthesis by rolling circle replication
(see textbook, Fig 19.4). After a complete + strand has been copied,
CisA re-ligates the DNA, creating a circular ssDNA + strand. Early in
infection, the replication cycle produces several RFs; late in infection,
the only product of replication is progeny + strands that are packaged
into viral capsids.
X174 is also notable for containing overlapping genes, a strategy
commonly used in viruses to maximize use of limited genome space.
One section of genomic material can specify the production of multiple
translational products by using different translational start sites within
the same mRNA with different reading frames.
An example of a lytic dsDNA virus is T4.
T4 contains linear dsDNA, approximately 170 kb in length. T4 genome
is about 85% identical to T2 and T4 phage, with differences relating to
receptor binding sites (T4 binds to LPS)
T4 overwhelms the host cell synthesis to enable its own proliferation
(Fig. 10.15/slide 16):
1. inhibits host RNA synthesis via ADP ribosylation of host RNA
pol sigma factor (inactivating the sigma factor); T4 makes its own sigma
factor that associates with core RNA pol for T4-specific transcription.
2. T4 encodes several genes for nucleases that degrade the host cell
DNA. T4 makes its own DNA pol, DNA ligase, etc. In addition, T4
DNA is modified so that the T4 DNA is resistant to the nucleases.
During T4 DNA replication, the newly synthesized phage DNA
undergoes recombination, forming long concatamers (linear molecules
of several genomes attached to one another; Fig. 10.13). The
concatamers are processed into pieces each about 170 kb in length,
representing one "headful" of DNA. This length is one genome plus
about 5000 bp present at both ends. Individual pieces that are packaged
into phage heads have different terminal repeats, but every piece has a
complete genome plus the duplicated sequence at the ends. This
arrangement is said to represent a "circular permutation" of the T4
genome.
In contrast, other dsDNA phages like T7 and lambda DNAs (which also
form concatamers during replication) are always cleaved at a particular
sequence in the genome, so that all the capsid-packaged linear dsDNA
molecules are the same (see Fig 19.7).
A different example of a dsDNA virus is Lambda ().
While we could spend many days talking about lambda, our focus in
M410 on lambda is on its ability to have two different sorts of
interactions with a host cell: it can infect and carry out a lytic replication
cycle or the lambda DNA can integrate into the host chromosome and
form a lysogen (Fig. 10.16 and Fig. 10.18). Lysogens are reasonably
stable associations between the integrated prophage (phage DNA) and
the host bacterial chromosome; however, the lysogen can be “induced,”
resulting in the lytic replication of more phage particles. Because of this
capacity to make the lysis/lysogeny choice, lambda is a temperate phage
(many known examples of temperate phages). In its simplest terms, the
lysis or lysogeny choice is determined by the relative abundance of two
lambda-encoded proteins in the cell: cI repressor and the Cro activator.
If Cro predominates, there will be expression of the lytic lambda
proteins (enabling phage DNA replication, expression of capsid
components, etc); if cI predominates, a lysogen will form (and the
expression of lytic phage replication genes will be repressed in the
lysogen).