Explain what happens in the viral replication cycle.
All viruses must recogize and attach to host cells. They bind to specific receptors (some are known. Others are specific)
At least part of the virus has to get through the membrane and/ or wall to enter the cells. For some viruses, the whole thing is in. For others, only the genome gets in.
Gene expression then occurs
The final step is release
Summarize in a list of steps the viral replication cycle.
- 1) binding to receptor
- 2) entry and uncoating
- 3) early gene expression
- 4) replication of viral genome
- 5) late gene expression
- 6) assembly of virions
- 7) exit
There are two sets of genes
Early genes and late genes
Early viral proteins do what
direct replicatoin of viral genomes
Some turn off host defenses, the host's ability to make more proteins, and turn on proteins that make copies of the viral genome
THIS IS THE CRITICAL EVENT! THE DIVIDING LINE!
The main thing that happens after early viral protein synthesis (early gene expression) is what?
the turn on of late mRNAs, which make late viral proteins, which package viral genomes and assemble virions
Explain in depth the final step
- There are many ways of getting out of the cell
- - some physically fill up the cell and cause lysis
- - some make holes in the cell for release
- - others utilize host machinery and basically get exocytosed
- - you can make new viruses without actually killing the cell
In general, what does a virus have/ need
- receptor binding protein
- a way to uncoat
- anti-host factors ( not always necessary)
- transcription factors (not always necessary)
What can be the minimum a virus can be?
capsid and polymerase
a group of viruses that are positive sense ssRNA viruses that infect bacteria
Discovered as a group in the late 1960s
Why was the discovery of RNA viruses a good thing?
it occurred around the time they were studying the genetic code. Effective for discovering how we use mRNA and RNA
Explain the simplicity of the RNA viruses.
they are the simplest, only encoding a total of four proteins
They have capsids (naked icosahedral viruses/ T=3 (60 x 3= 180) and one maturation protein (on the surface of the virus and binds the genetic material)
They also have an A protein
They can be crystallized because they are inert
an ssRNA genome bound by a maturation protein
Diameter of 26 nm
What is the genome of RNA phages
3.5-4 kb in length; very simple
- Four proteins encoded in this order
- 1) maturation protein: binds the RNA; gives some structure; located outside
- 2) coat protein: cap protein; gives virus its structure
- 3) lysis protein
- 4) replicase: makes copies of the genetic material
RNA phages bind to F+ bacteria, which make the F pilus
They attach to the pilus via the maturation protein, which triggers an event in the pilus. The maturation protein is cleaved, releasing the RNA, which enters
What is the replication cycle of MS2 (ssRNA phage)
- 1) bind to the pilus
- 2) move down the cell wall
- 3) degradation of maturation protein (cleavage)
- 4) release of RNA
- 5) phage protein synthesis
- 6) release
What is the idea for how the RNA gets inside of the cell?
it is believed that the pilus that the RNA is attached to retracts into the cell dragging the RNA and cleaved maturation protein along with it.
Why is protein synthesis highly regulated?
1) different amounts of each protein are needed. Ex: for every 180 copies of the coat protein, the phage needs only 1 copy of the maturation protein
2) replication and translation of the same RNA molecule can lead to problems. Replication starts at the 3' end of the RNA and proceeds toward the 5' end. Translation occurs in the opposite direction. If improperly arranged, the replicase and ribosome would meet somewhere on the RNA and sit facing each other forever.
To regulate translation, what is the case?
access of ribosomes to the AUG start codons is strongly restricted. In fact, only the coat gene translational start site is able to bind directly to ribosomes.
This access is restricted by the secondary structure of RNA due to base pairing, which provides the control. The structure makes it only possible for the AUG codon to bind.
What are the forces that drive the binding of ribosomes to translational start regions on prokaryotic messenger RNAs?
1) base complementarity between the 16S ribosomal RNA and the Shine-Dalgarno sequence upstream of the start codon
- 2) interaction of the anticodon on the initiator fmet-tRNA with the AUG start codon on the messenger RNA
- 3) binding of rbosomal protien S1 to pyrimidine-rich sequences frequently found upstream of the Shine-Dalgarno sequence
If a strong secondary structure in mRNA prevents interaction with one of the above components, what/
ribosomes will not efficiently bind and therefore protein synthesis will not begin at that site
Secondary structure in RNA consists of regions of self-complementary base sequence that form stem-loop structures.
What are the three sites of mRNA that can prevent the ribosome from binding?
- 1) stem MJ
- 2) the operator hairpin containing AUG itself
- 3) stem R32
Explain what happens as soon as the ribosome finds AUG.
- it starts making coat proteins. The others are produced later
- As you start making the coat protein, the structure of RNA is starting to change
The ribosome, as it moves, breaks the base pairs apart, releasing a second start codon for the replicase
This is temporal organization. The next thing that happpens is synthesis of the polymerase to start making viral genomes
What is a second level of control for the translation of replicase?
when the concentration of coat proteins becomes sufficiently high in the cell, dimers are formed. These dimers bind to the operator hairpin, recluding furter trnaslational starts of the replicase gene
In other words, ribosomes translating the coat gene disrupt secondary stucture, allowing replicase translation
Ribosomes terminating coat translation can do what?
reinitiate at the lysis gene start site
What is the replication versus translation thing going on?
competition for the same RNA template
Replicase prtein binds to RNA and competes with ribosome
- Recplication proceeds if replicase binds first
- - minus strand copies of the plus stranded genome are made
- - minus strands are then copied into plus strands
The ribosomes can make ___ after it reaches the __. But, this occurs at a smaller rate than the __.
It can also just __ or __.
- replicase protein
- coat termination point
- just fall off
- walk back the other way and hit the lysis start codon.
Explain protein synthesis in the order in which it occurs?
- coat is made immediately
- coat translation makes replicase and lysis accessible
- maturation protein needs to be made last
How can replicase act as a negative regulator for a ribosome?
- it has the capability of binding at the first coat start site
- It acts as not only a polymerase but also as a TF, preventing more ribosomes from binding
It copies the RNA into complementary minus sense strands, which is a template to make several more copies of the RNA
Once the replicase s made, what can occur?
It can begin to generate new copies of phage RNA.
The plus strand genome is first copied into a complementary minus strand RNA
this RNA is in turn used as a template to produce more plus strand RNAs.
How does the replicase copy the genome RNA starting at its 3' end while ribosomes are translating the same RNA in the opposite direction?
Replicase binds to the RNA at two internal positions, one of which is the start of the coat gene. As a result, there is competition between replicase and ribosomes for this site. If the replicase arrives first, there will be no new translation, allowing replicase to copy the RNA with no oncoming traffic. On the other hand, if the ribosome binds first to the coat proteing ene, replicase will not be able to bind to its template, and therefore the ribosome is free to complete its voyage unhindered
What does the maturation protein do?
puts it in the capsid. It is made last.
It is the only late gene in the virs
What is the trigger that keeps the protein from being made until the end?
it starts off as inaccessible due to base pairing
Bacterial ribosomes find the shine-Delgarno sequence
The maturation protien can't get made by genetic material of the virus once it gets into the cell
The virus material that gets into the cell is never in teh form that allows the maturation start codon to be accessible. ONce you start making other genes, an opportuniy is presented to allow the maturation protiens to be made.
What is the window of opportunity that allows the maturation protein to be synthesized?
When the RNA is first coming out of the poylmerase, it won't refold into a cloverleaf sequence right away. The temporary state that it is in allows the maturation protein to be synthesized.
What are the end steps?
- Maturation protein binds to RNA and they form a place for the capsid protein to come together (self-assembly) into a completed virus (coat protein forms icosahedral shell around RNA)
- accumulation of lysis proteins results in collapse of cell wall
What is the big picture?
- these phages are models for the simplicity of some viruses
- 1) four proteins
- 2) no room for promoters, TFs, etc. There are alternate mechanisms for gene regulation. They can still be turned on and off. But, you can steal genes from the host. The virus doesn't need to make them
- 3) Uses host genes
What can we learn about protein amounts from these steps?
coat protein is the most abundant protein in the virus; it is the easiest to make and gets made the most
The intermediate proteins are replicase and lysis proteins
The maturation protein is the last to be made (ratio of coat protein to maturation is 180:1; there is one small mechanism that turns on briefly to turn on the maturation protein
You end up with the right amount of proteins in the cell to make functional virus particles. There is no scaffolding protein nor are there quality control steps.
What is the final step?
the final step is accumulation of the lysis protein in the cytoplasmic membrane has caused the collapse of hte cell wall.