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 fX174.

The example of an iscosahedral ssDNA phage is fX174.

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.

fX174 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 (l).

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).