Bacterial viruses contain either a ssRNA, ssDNA, or a dsDNA genome (rarely a dsRNA genome) (Fig. 9.12). 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). Base pairing interactions only occur between a positive and a negative strand. A positive strand ssRNA virus has a genome that acts as mRNA immediately after entering the cytoplasm of the infected host cell. (Fig. 9.11)

The morphology of the viral capsids can be filamentous, icosahedral, or prolate icosahedral with helical tails. While viruses of eukaryotes can be either naked or enveloped, most bacteriophages are naked (without membrane envelopes).

A typical lytic phage goes through a replication cycle 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. (Fig. 9.8)

The example described in class of a ssRNA phage is MS2 (closely related to phage Qb). (Fig. 16.1 & 16.2)

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). (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 described in class of a filamentous ssDNA phage is M13. (Fig. 16.5)

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 lysing/killing the host cell. The phage capsids are assembled as the virus "buds" from the cell, allowing continued growth of the host.

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, described below.

The example of an iscosahedral ssDNA phage is fX174. (Fig. 16.3 & 16.4)

circular, + ssDNA, 5368 nucleotides in length, encoding 10 genes.

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. After 1 complete + strand has been copied, CisA re-ligates the DNA, creating a circular ssDNA + strand and a dsDNA. Early in infection, the replication cycle produces several RFs; late in infection, the only product of replication is progeny + strands that associate with other viral proteins and are packaged into viral capsids.

fX174 is also an example of overlapping genes. One section of genomic material can specify the production of multiple translational products by:

1. translation of 1 gene from 2 different translational initiation sites (example: CisA/CisA*)--the amino acid sequence of CisA* is the same as the C-terminus of CisA

2. translation of 2 different genes from the same section of mRNA in 2 different translational reading frames (example: CisA and CisB).

The example of a lytic dsDNA virus discussed in class is T4. (also look at the text about T7 pp 507-510)

T4 contains linear dsDNA, approximately 165 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; see Fig. 9.10)

T4 overwhelms the host cell synthesis to enable its own proliferation: (Fig. 9.15)

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. (In contrast, T7 makes its own RNA polymerase, by-passing the need for the host cell RNA pol during late viral infection)

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. This modification is on the cytosine bases of the DNA, which are in the form of 5-hmC; a further modification--glucosylation-- is added post-replication. (Fig. 9.14)

During T4 DNA replication, the newly synthesized phage DNA undergoes recombination, forming long concatamers (linear molecules of several genomes attached to one another). 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. (Fig. 9.13)

In contrast, 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.

Phage lamda (l) and temperate bacteriophage will be discussed as part of the transduction lecture.

Also, look at short section on Archaeal phage on pp. 512-513.

 

revised 11/23/05