Double-Stranded DNA Viruses

Double-Stranded DNA Viruses Infect All Cell Types

Perhaps the largest group of known viruses is the double-stranded (ds) DNA viruses; most bacteriophages have dsDNA genomes, as do several insect viruses and a number of important vertebrate viruses, including herpesviruses and poxviruses. The pattern of multiplication for dsDNA viruses is shown in the figure.

The synthesis of DNA and RNA is similar to what occurs in cellular organisms; therefore some dsDNA viruses can rely entirely on their host’s DNA and RNA polymerases.

Bacteriophage T4: A Virulent Bacteriophage

The life cycle of T4 bacteriophage (family Myoviridae, species Enterobacteria phage T4) serves as our example of a virulent dsDNA

Multiplication Strategy of Double Stranded DNA Viruses
Multiplication Strategy of Double-Stranded DNA Viruses
Because the genome of dsDNA viruses is similar to that of the host, genome
 replication and mRNA synthesis closely resemble that of the host cell and can
 involve host polymerases, viral polymerases, or both. The DNA genome
 serves as the template for DNA replication and mRNA synthesis. Translation
 of the mRNA by the host cell’s translation machinery yields viral proteins,
 which are assembled with the viral DNA to make mature virions. These are
 eventually released from the host.

phage. Virulent (lytic) bacteriophages are capable only of the lytic cycle. That is, their infection of a host always ends with cell lysis. As with most viruses (except plant viruses), the first step of viral infection is attachment (adsorption) to the host cell surface.

T4 attachment begins when a tail fiber contacts either the lipopolysaccharide or certain proteins in the outer membrane of its Escherichia coli host (figure). As more tail fibers make contact, the baseplate settles down on the surface (figure). After the baseplate is seated firmly on the cell surface, the baseplate and sheath change shape, and the tail sheath reorganizes so that it shortens from a cylinder 24 rings long to only 12 rings (figure).

As the sheath becomes shorter and wider, the central tube located within the sheath is pushed through the bacterial cell wall. The baseplate contains the protein gp5, which has lysozyme activity. Lysozyme is an enzyme that breaks the bonds linking the sugars of peptidoglycan together (see figure). Thus this protein aids in the penetration of the tube through the peptidoglycan layer.

Finally, the linear DNA is extruded from the head, probably through the central tube, and into the host cell (figure). The tube may interact with the plasma membrane to form a pore through which DNA passes. Within 2 minutes after the entry of T4 DNA into an E. coli cell, the E. coli RNA polymerase starts synthesizing T4 mRNA (figure).

This mRNA is called early mRNA because it is made before viral DNA is made. Within 5 minutes, viral DNA synthesis commences, catalyzed by a virus-encoded DNA-dependent DNA polymerase. DNA replication is initiated from several origins of replication and proceeds bidirectionally from each. Viral DNA replication is followed by the synthesis of late mRNAs, which are important in the later stages of the infection.

As with most viruses, the expression of T4 genes is carefully timed, and the transcripts and protein products of the genes are named to reflect the time of appearance. Thus depending on the virus, terms such as immediate-early, middle, and late may be used. T4 controls the expression of its genes by regulating the activity of the E. coli RNA polymerase.

Initially, T4 genes are transcribed by the host RNA polymerase and the housekeeping sigma factor s70 (see table).

T4 Phage Adsorption and DNA Entry
T4 Phage Adsorption and DNA Entry
Adsorption and viral DNA entry into the E. coli host are mediated by the phage’s tail fibers and base plate. (f) An electron micrograph of an E. coli cell being infected by T-even phages. These phages have released their dsDNA into the cell and now have empty capsids.
MICRO INQUIRY What enzyme found in the T4 baseplate facilitates penetration through the cell wall?
The Life Cycle of Bacteriophage T4
The Life Cycle of Bacteriophage T4.
A diagram depicting the life cycle with the minutes after DNA ejection given for each stage.
MICRO INQUIRY Why do you think T4 evolved to initiate DNA replication from multiple origins, rather than from a single origin of replication as seen in its host cell?
5 Hydroxymethylcytosine HMC
In T4 DNA, the HMC often has glucose attached to its hydroxyl.

catalyzes addition of the chemical group ADP-ribose to one of the a-subunits of RNA polymerase (see figure). This modification helps inhibit the transcription of host genes and promotes viral gene expression. Later the second a-subunit receives an ADPribosyl group.

This halts transcription of some early T4 genes but not before the product of one early gene (motA) stimulates transcription of somewhat later genes. One of these later genes encodes the sigma factor gp55.

This viral sigma factor helps the host cell’s RNA polymerase core enzyme bind to viral late promoters and transcribe the late genes, around 10 to 12 minutes after infection.

Bacteria combine several regulatory mechanisms to control complex cellular processes (section)

The Terminally Redundant Circularly
The Terminally Redundant, Circularly
Permuted Genome of T4.

The tight regulation of T4 gene expression is aided by the organization of the T4 genome, in which genes with related functions—such as the genes for phage head or tail fiber construction—are usually clustered together.

Early and late genes also are clustered separately on the genome; they are even transcribed in different directions. A considerable portion of the T4 genome encodes products needed for its replication, including all the protein subunits of its replisome and enzymes needed to prepare for DNA synthesis.

Some of these enzymes synthesize an important component of T4 DNA, hydroxymethylcytosine (HMC) (figure). HMC is a modified nucleotide that replaces cytosine in T4 DNA. Once HMC is synthesized, replication ensues by a mechanism similar to that seen in bacteria.

After T4 DNA has been synthesized, it is modified by the addition of glucose (a process called glycosylation) to the HMC residues. Glycosylated HMC residues protect T4 DNA from attack by E. coli endonucleases called restriction enzymes, which would otherwise cleave the viral DNA and destroy it.

This bacterial defense mechanism is called restriction. Other phages also chemically modify their DNA to protect against host restriction. Restriction enzymes (section) The linear dsDNA genome of T4 is generated in an interesting manner involving the formation of long DNA molecules called concatemers, which are composed of several genome units linked together in the same orientation (figure).

How does this occur? As we discuss in chapters 13 and 15, the ends of linear DNA molecules cannot be replicated without mechanisms such as the enzyme telomerase. T4 and its E. coli host do not have telomerase enzyme activity. Therefore each progeny viral DNA molecule has singlestranded 39 ends.

These ends participate in homologous recombination with doublestranded regions of other progeny DNA molecules, generating concatemers. During assembly, concatemers are cleaved such that the genome packaged in the capsid is slightly longer than the T4 gene set.

Thus each progeny virus has a genome unit that begins with a different gene and ends with the same set of genes. If each genome of the progeny viruses were circularized, the sequence of genes in each virion would be the same.

Therefore the T4 genome is said to be terminally redundant and circularly permuted, and the genetic map of T4 is drawn as a circular molecule. The formation of new T4 phage particles is an exceptionally complex self-assembly process that involves viral proteins and some host cell factors (see figure).

Late mRNA transcription begins about 9 minutes after entry of T4 DNA into E. coli (figure). Late mRNA products include phage structural proteins, proteins that help with phage assembly but do not become part of the virion, and proteins involved in cell lysis and phage release.

These proteins are used in four fairly independent subassembly lines that ultimately converge to generate a mature T4 virion. A critical step in T4 virion construction is filling the head portion of the virion with the T4 genome. This is no simple matter—the dsDNA genome is somewhat rigid and has many negatively charged moieties.

Therefore the dsDNA must essentially be crammed into the developing head. This is accomplished by a complex of proteins sometimes called the “packasome.” The T4 packosome has more power than an automobile engine. The packasome includes a protein called terminase, which has two functions: to cut the concatemers formed during T4 genome replication and to push the DNA into the T4 head.

For each head filled, it uses these functions in the order “cut, push, cut.” The first cut generates a double-stranded end on a concatemer. This end is then threaded into the portal, as terminase pushes the DNA into the head, using energy supplied by ATP hydrolysis, until the phage head is filled with DNA—a DNA molecule roughly 3% longer than the length of one set of T4 genes.

Terminase then makes its second cut, and the packaging process for that head is complete. Terminase then leaves the head, and several other viral proteins bind the head at the portal through which the DNA entered. This seals the head and prepares it for addition of the tail and tail fibers.

Finally, virions are released so that they can infect new cells and begin the cycle anew. T4 lyses E. coli when about 150 virus particles have accumulated in the host cell. T4 encodes two proteins to accomplish this. The first, called holin, creates holes in the E. coli plasma membrane.

The second, an endolysin called T4 lysozyme, degrades peptidoglycan in the host’s cell wall. Thus the activity of holin enables T4 lysozyme to move from the cytoplasm to the peptidoglycan so that both the plasma membrane and the cell wall are destroyed.

Key Concepts

Double-Stranded DNA Viruses Infect All Cell Types

■ T4 is a virulent bacteriophage that causes lytic infections of E. coli. After attachment to a specific receptor site on the bacterial surface, T4 releases its dsDNA into the cell (figures). T4 DNA contains hydroxymethylcytosine (HMC) in place of cytosine, and glucose is often added to the HMC to protect the phage DNA from attack by host restriction enzymes (figure). T4 DNA replication produces concatemers, long strands of several genome copies linked together (figure).

■ Lambda (l) phage is a temperate bacteriophage. It can establish lysogeny, rather than pursuing a lytic infection. During lysogeny, the viral DNA, called a prophage, is replicated as the cell’s genome is replicated. Lysogeny is reversible, and the prophage can be induced to become active again and lyse its host. This highly regulated process is an important model system for regulatory processes. The protein cII plays a central role in regulating the choice between lysogeny and a lytic cycle. If ceII protein levels are high enough, lysogeny is established. If not, the lytic cycle is initiated (figures).

■ Less is known about archaeal viruses. However, many of those discovered have interesting morphologies, which has led to the creation of several new viral families (figure 27.12). Many establish chronic infections, whereas others establish lysogenic and lytic infections (figure).

■ The CRISPR/Cas system is a set of sequences found in many archaeal and bacterial genomes (figure). It is a defense system that protects the cells from viral attack. It functions in a manner similar to RNA silencing, an antiviral defense mechanism observed in eukaryotic cells.

■ Herpesviruses are a large group of dsDNA viruses. They cause acute infections such as cold sores, genital herpes, chickenpox, and mononucleosis. This is followed by a lifelong latent infection in which the virus genome resides within neurons. The virus can be reactivated at later times to cause a productive infection.

■ Nucleocytoplasmic large DNA (NCLD) viruses, also called megaviruses, are members of several virus families. They are thought to share an evolutionary history and are interesting due to their large genome and virion size. Furthermore, their genomes contain genes for translation-related functions that are not usually observed in viral genomes.

Leave a Comment