DNA replication, genomic RNA synthesis, and protein sysnthesis within a cell are simple, compared to these processes within the viral life cycle. When considering how viruses replicate, there are problems associated with the fact that viral genomes, even if they are DNA, are not necessarily linear and double-stranded like chromosomal DNA, that the infectious cycle is temporally regulated, so that gene expression and genome replication must be coordinated, and the the virus must insure that its own proteins are produced.
DNA replication, genomic RNA synthesis, and protein sysnthesis within a cell are simple, compared to these processes within the viral life cycle. During this class meeting, we will be discussing the mechanisms that viruses use to overcome the problems associated with
- the fact that viral genomes, even if they are DNA, are not necessarily linear and double-stranded like chromosomal DNA,
- that the infectious cycle is temporally regulated, so that gene expression and genome replication must be coordinated,
- that the the virus must insure that its own proteins are produced at the expense of the cellular proteins.
DNA synthesis
The problems
Within a eukaryotic cell, chromosomal DNA is replicated by a set of enzymatic reaction that have been well characterized. One major problem with such replication is what happens at the 5' end of the newly synthesized strand. What type of molecule is at that end? What problems result from having that molecule within a chromosome? How do nuclear chromosomes deal with this problem?
Cellular DNA synthesis in prokaryotes proceeds essentially throughout the cell cycle. Within eukaryotic cells, synthesis is not continuous; the time from one cell division to the next can be broken into phases - G1, S, G2, M. Moving from one phase to another is highly controlled - for example, entrance into S requires passing "start", a set of molecular signals that are over-riden if the chromosomal DNA is damaged. Viral DNA enters the cell - how does it deal with the fact that cellular DNA synthesis occurs during a short portion of the cell cycle?
The solution to the 5' primer problem
To answer these questions - since all DNA synthesis requires a primer, the 5' end of the new strand is RNA. This means that not all of the replicated strand is DNA. This can't be. To overcome this problem chromosomes repair this RNA by use of an enzyme called telomerase (the end of the chromosome is called a telomere.) Parenthetically, it is believed that aging is caused in part by incoplete repair of the ends,
Viral chromosomes do not have telomeres, so how do they solve the problem of the primer? Actually very simply;
To have a primer but not an end, the viral genome is circular. The RNA is incorporated into the newly synthesized strand, but since the DNA is circular, there is no difficulty with the ongoing synthesis excising the RNA and replacing it with DNA.
- they have an RNA primer, but they don't have an end
- they use something other than RNA as the primer.
- Can you imagine what the situation is in each of these cases?
You may think that the notion of a primer other than RNA is unusual, but if we think for a moment about the minimum requirement for priming, it may become clearer. All that is really needed for initiation of DNA synthesis is a 3' OH. What other biological molecule could provide this group? Yes - it is an amino acid. What are the amino acids that have hydroxyl groups on their side chains? If a protein is used as primer, what is found at the end of the newly synthesized strand?
One example of a virus that uses protein priming is Adenovirus (Ad). Ad is a DNA virus with a linear double-stranded genome of about 35 kbp. Within the virion, the virion has a 55 kDa 5' terminal protein. The protein is a cleavage product of the 80 kDa protein that is the actual protein used for priming. This cleavage occurs during the maturation of the virus.
One difficulty with protein priming is how does the protein actually find the template for synthesis of viral DNA? Not only does the primer have to find the template, but it has to find the end of the template. For Ad, the answer may not seem satisfactory, but it seems to work - which is good enough. The 3' terminal nucleotide is a G. A reaction between the 80 kDa protein and cCTP results in the formation of a phosphoester bond through a hydroxyl on a serine within the protein. This C lines up with the G on the template. The replication occurs using a virally coded DNA polymerase. The localization of replication initiation at the ends of the viral DNA is facilitated by the identity of the nucleotides between 9 and 18, counting in from the ends, that also bind the polymerase.
Take a look at figure 17.1
Another virus that uses protein as a primer is Hepatitis B virus (HBV). HBV has a double-stranded circular genome. Try to figure out how HBV copes with both a circular genome and a protein primer.
The solution to the timing of DNA synthesis problem
When viral DNA enters the cell - how does it deal with the fact that cellular DNA synthesis occurs during a short portion of the cell cycle? If the genome has to sit and wait for S phase, it seems hard to imagine how the virus can take over the cell's synthetic machinery as efficiently as it does. Can you think of a way(s) that the virus can overcome this problem?
One way is for the virus to encode its own DNA polymerase, as do Ad and HBV. Under these circumstances, viral DNA synthesis is disconnected from cellular DNA synthesis.
Other viruses just give up on this problem and use the cellular DNA synthetic machinery to replicate its DNA. A consequence of using the cell's machinery is that viral DNA replication occurs only during S-phase. One group of parvoviruses, the autonomous parvoviruses, use the cell's DNA polymerase to replicate its single-stranded DNA genome. One reason for this is that their genomes are too small to code for a polymerase.
Other viruses that use the cell's DNA synthetic apparatus aren't as wimpy as the parvoviruses. SV40, a double-stranded DNA virus, infects monkey cells. Upon infection, the protein product (T antigen) of an early gene, causes the cell to enter the S-phase of the cell cycle. T antigen, interacts with a cell coded protein called p53 and inactivates it. p53 is one of the proteins that is a gate-keeper at "start". Once p53 can't carry out this function, DNA synthesis occurs.
We have previously discussed the fact that for viruses with (-) RNA genomes, a complementary (+) strand is produced on the template of the genomic (-) strand. This (+) strand is the template for production of the (-) RNA genomes that are encapsidated. On the other hand, the genomic (-) strand is also the template for synthesis of mRNA. Can you imagine how this could give rise to a difficulty?
If the mRNAs are shorter than genome length, two different size classes of (+) RNA must be produced; the mRNAs as well as the full-length (+) to serve as template for the full length (-). How is this accomplished?
Vesicular stomatitis virus has an interesting mechanism for doing this.
The diagram, figure 17.2 shows the genome of VSV and the names of the genes that it encodes. It also shows the structure of the complete virion. At the very left end of the genome is a 48 nucleotide leader sequence - a set of nucleotides that is not translated into protein. This leader is one of two central elements in the switch between mRNA synthesis and the synthesis of the full-length (+) to act as template for the synthesis of progeny genomes. The other element is protein N.
mRNA synthesis begins at the leader, at the left end of the genome. Synthesis proceeds past the leader and into the N gene. The leader does not remain attached to the N gene mRNA. Instead, the 5' end of the mRNA for N protein becomes capped. At the 3' end of the N gene is a novel method for polyadenylation. In between each of the genes on the VSV genome is a stretch of seven Us. These Us are the template for the synthesis of the poly A tail. The replicase "stutters" over these seven Us, using them repetititely as template to produce the tail. After the first mRNA is complete, synthesis of the mRNA for the next gene (NS) begins. It is not clear if the replicase falls off the genome or if transcription proceeds into the NS gene and there is a cleavage event that separates the two mRNAs.
The switch to synthesis of full-length (+) is regulated by the concentration of N protein. As the concentration increases, binding of N to the leader occurs. This binding results in the leader remaining attached to the downstream sequences for N; the replicase just continues synthesizing to the end of the template.
Taking over the cell's machinery
One of the central elements of viral replication is the use of the cellular ribosomes to produce viral proteins rather than cellular proteins. How is this accomplished? Since the genomes of viruses are smaller than the complete array of chromosomal genes, there is a greater mass of viral mRNA produced during infection, that can compete efficiently for access to the protein synthesizing machinery. However, polio virus has developed a novel mechanism to insure that its mRNAs get translated while cellular mRNAs do not.
Polio virus mRNA is as long as the genome. The mRNA is translated as one long polypeptide, what we call a polyprotein, that is cleaved into its consitutent, smaller proteins. This cleavage occurs even as the polyprotein is being produced - one of the proteins coded by the genome has this protease activity. Polio virus is one of those viruses that has a (very small) protein at its 5' end. This protein is call Vpg and is only 27 amino acids long. It is bound to the RNA through a tyrosine residue. (NOTE: this protein is NOT required for priming! ENZYMES THAT PRODUCE RNA, whether the polymerases that produce mRNA from a DNA template or the viral replicases that produce RNA from an RNA template, DO NOT REQUIRE A PRIMER.) Having this protein on the 5' end means that the poliovirus mRNA does not have a 5' cap. This difference from cellular mRNA is the basis for polio's taking over the cell's protein synthesis. Can you come up with a hypothesis for how this might occur?
The 5' cap on cellular mRNAs is recognized by the ribosome. If the protein(s) that recognize this cap were destroyed, then the ribosomes would not be able to recognize the cap and cellular mRNAs would not be translated. However, since polio mRNAs do not ever have a cap, they alone would be translated in infected cells.
DNA replication, genomic RNA synthesis, and protein sysnthesis within a cell are simple, compared to these processes within the viral life cycle. When considering how viruses replicate, there are problems associated with the fact that viral genomes, even if they are DNA, are not necessarily linear and double-stranded like chromosomal DNA. The difficulty arises from the RNA nature of the primer for DNA synthesis. With short, linear chromosomes, how can the primer be converted into DNA? One mechanism is to have circular genomes, so that DNA synthesis can keep going around the circle, eliminating the primer and converting it to DNA. Another mechanism used by viruses is to eliminate RNA primers entirely, and use protein as primer. Although this sounds peculiar, it makes sense in light of the fact that the minimnal requirement for priming is a 3'-OH.
For single-stranded (-) RNA viruses, two types of (+) RNA must be produced. One type is mRNA, that is shorter than geome length, and the other type is the full length (+) strand, that is the required template for the synthesis of full length (-) progeny genomes. VSV uses a complicated mechanism, based on the concentration of a virion structural protein, to regulate the type of (+) RNA produced.
Poliovirus insures the synthesis of its proteins at the expense of the translation of cellular mRNAs by altering the transcriptional apparatus to suit the characteristics of polio mRNA. Viral mRNA is not capped, so the virus destroys the ability of the ribosomes to recognize cellular mRNA, which is capped. As a result, the only mRNAs that can be translated are viral.