Flashcards in SH5-6: Introduction to viruses and virus structure Deck (22)
What is meant by 'obligate intracellular parasite' and why do we refer to viruses as this?
It is used to show that viruses can only grow inside a host cell. All viruses that we know of lack all the necessary information required to make proteins and ATP. The host cell provides these mechanisms.
What are the groups for viral classification (smallest to biggest)?
• Quasi species (RNA viruses only ), Strain/type, Genus, Family, Order
What is the average size of a virus?
20-400nM (very small)
What was the classification of viruses originally based on?
Morphology - size, shape, enveloped/undeveloped. Physiochemical properties - molecular mass, thermal stability, ionic stability.
What is the classification of viruses based on now?
Genome - RNA, DNA, segmented sequence. Sequence macromolecules - protein composition and function.
Why do we look at the whole genome of a virus when classifying/ looking a their phylogeny?
As no one gene is universal to viruses, unlike bacteria or eukaryotes
What is the regressive theory for virus origins?
Viruses were larger organisms, but reduced in size, losing the ability to carry out functions/mechanisms. This is not a popular theory
What is the progressive theory for virus origins?
Normal cellular nucleic acids gained the ability to replicated on their own. DNA viruses came from plasmid type elements. Retroviruses came from retrotransposons. RNA came from mRNA.
What is the co-evolution theory for virus origins?
Viruses co-evolved with cellular life
How can we study viruses?
• Living hosts/ Animals (unethical)
• Embryonated/ fertalised eggs
• Organ cultures or primary cell cultures- taking a cell sample from a human, culture and replicated. (cells/organs of each person differ)
• Immortalized cell lines- populations of cells that are clonal, they have one origin and have the ability to grow in the lab indefinitely. Properties remain relatively constant. (Currently the most popular method)
How can we count/ quantify viruses?
• Plaque assay- grow dilution of virus on agar, the number of plaque relates to the number of viral particles. Counted in PFU’s (plaque forming units) or CFU’s (units)
• Directing numeration- electron microscope used to count viral particles of an appropriate dilution on a grid
• Quantative PCR- determines the number of genome (1 genome= 1 virus particle)
• Proteins the virus is expressing
• Serology- counting antigens of the virus in the blood
• Structural studies- enrich and purify the viruses, x-ray crystallography, electron microscopy
• Biochemical techniques- look at the genome of the virus (size, RNA, DNA)
• Genetic techniques- looking at sequence, variations and the effect the variations have on the phenotype of the virus
• Molecular biology techniques- nucleic acid sequencing, changing the genome and seeing how it effect he replication in a host cell
• Chemistry- Looking at the polo virus which is RNA. Convert to DNA sequence by taking oligonucleotides and assemble them using DNA ligase to form the DNA genome. At the 5’ end stuck the promoter element for and enzyme called t7RNA polymerase (this is a virus enzyme which will copy the DNA into RNA). Add this synthetic RNA form of polio to a lysate of HeLa cells (mammalian cells). Take the mixture, then add them to cells on a plate and it shows varium particles formed in the lysate, and they are being amplified in the cells.
What are the two genetic methods used to determine the structures of a virus?
X-ray crystallography and electron microscopy
Describe electron microscopy.
Until recently you couldn’t resolve atom structures from this method, but has been resolved so you can now determine the structure. Take the sample of the virus and flash freeze it (rapidly freeze) with liquid nitrogen, this causes the water in the structure to stay in a liquid state, so the macromolecules stay hydrated and do not change shape. Use you electron microscope to shine an electron beam through the sample, and capture pictures on a detector, and you can then determine the 3D structure of the sample.
Describe X-ray crystallography.
Crystallise virus sample, fire beam of x-rays at it, get a diffraction pattern which is picked up by an x-ray detector. This pattern helps us deduce the 3D structure. Some viruses don’t form crystals so this method doesn’t work
What are X-ray crystallography and electron microscopy used together to do?
Build up the atomic resolution structures of viruses. From this you can design drugs against the virus
What is a capsid?
A protein layer surrounding the genome of the virus
What is the helical structure of a capsid?
This is a simple way to arrange multiple identical subunits. The use of rotational symmetry to arrange irregularly shaped proteins (triangular) around the circumference of circle to form a disc. You stack multiple discs to form a cylinder. The virus is formed by these protein discs and the RNA genome which sits inside the discs. The capsid does not consist of the discs, it consists of the proteins in a helical symmetry, and this is because the nucleic acids have a helical symmetry. When you introduce the helical nucleic acids to the radial structure (discs), it binds to the interior of the core of the radial structure and changes the configuration to a helical arrangement. More radial structures are added, and become helical when the nucleic acids are added. Nucleic acid is draw up the capsid by electrostatic interactions (between negatively charged nucleic acids and positively charged proteins).
What is the icosahedral structure of a capsid?
Proteins form a hollow spherical structure enclosing the genome within. Formed from 20 equilateral triangles angled into a sphere- these equilateral triangles can be one subunit or made up of multiple subunits.
Bacteriophage QX174 has 60 identical subunits, with 3 subunits forming one triangle. T1 viruses have 60 subunits, T3 have 180 subunits, T4 240 subunits. In T1 the 3 subunits making the equilateral triangle are perfectly symmetrical. In other T forms, the subunits are not perfectly symmetrical, so they are given the name ‘quasi symmetry’ (roughly, but not perfectly symmetrical).
T3 quasi equivalence (180 subunits) can form slightly different structures within the equivalent triangle. In one form, you get dimers/ capsomers that form between the triangles (so 2 triangles are closely related)- this makes 90 dimers/ capsomers. In another form, you get 60 trimers/ capsomers where 3 triangles are closely related to each other. The last form gives you 12 pentamers as 5 triangles are closely related to each other.
Biochemically fractionating these forms with either give you dimers, trimers or pentamers, and then you can identify the capsid structure.
Has 2-3-5 (2-fold 3-fold 5-fold) rotational symmetry.
What are the major function of the capsid?
• Protect the fragile nucleic acid genome
• Physical damage- shearing/ fragmenting the genome
• Chemical damage- sunlight destroys by UV irradiation leading to chemical modification, enzymatic damage.
• Responsible for getting the virus particle into a new cell for reproduction. Capsid must facilitate the entry into a new cell, does this with binding a specific virus-attachment protein (often called an anti-receptor) to a cellular receptor molecule
Why do capsids exists in subunits?
• This structure is most energetically favourable. This is proved by an experiment carried out in 1955, with pure tobacco mosaic virus RNA and coat protein. When put together the virus particle spontaneously forms with subunits. This is because the virus is in a minimum free energy state. It is thermodynamically the most stable structure for the virus, so entropy drives the formation of this structure.
• Protein subunits in a virus capsid are multiply redundant (many copies per particle). If you damage one subunit it does not always destroy the others.
• Nucleic acids only code for 15% of its weight as proteins, but we know viruses are composed of 50-90% by weight of proteins, so it must repeat itself, giving the subunits.
• Assembly of identical subunits is easier. Smaller protein/gene means less chance of error in making subunits, if there is an error, less waste. Larger number of subunits, more stable the virus.
What is an envelope?
This is a lipid bilayer that only some viruses have. Many acquire this lipid bilayer when they come through the host cell from the Golgi apparatus or the endoplasmic reticulum.