Virus Basics
Virus Shapes
Helical
Slinky-shaped capsid that twists around and encloses its genetic material
Polyhedral
Genetic material surrounded by a many-sided capsid
Spherical
Helical viruses enclosed in a envelope, spiked with sugary proteins that assist with attachment & entry
Complex
Polyhedral head, helical body (tail sheath), & legs (tail fiber) that attach to a cell membrane to directly inject its genetic material
Why Do We Care About Viruses?
The number of viruses on Earth is staggering
More viruses in a liter of coastal water than people on Earth
1030 bacteriophages in worlds waters (a phage weighs 10-15 grams; 1030 × 10-15g exceeds the weight of all elephants on the planet by 1000x). If you put 1030 phages end to end it would equal 100 million light years
Whales excrete 1013 calciviruses a day in feces. These viruses can cause blisters, rashes and GI complications in other mammals
Approximately 1016 HIV genomes exist on the Earth today
How infected are we?
90% of people are infected with HSV-1, HSV-2, VSV, HCMV, EBV, HHV-6, HHV-7, HHV-8
Once infected, it’s for life!
We regularly eat and breathe viral particles (many not being infectious)
Some viruses are beneficial to life on this planet
Ability of grass to thrive near hot springs is dependent upon virus that infects fungus that infects the plant. All are needed to provide tolerance
Parasitic wasps lay eggs in caterpillar. Releases polydnavirus with the egg, and the virus immunosuppresses caterpillar so it won’t reject the eggs
Viruses were First Identified as “Filterable Agents” Capable of Causing Disease
Virus: latin for poison, slimy liquid
General term for any liquid that would make one ill
Louis Pasteur used the filterable agent concept to isolate rabies (although convinced that it was bacteria, not a virus)
Beijerinck attempted to give scientific name for viruses: contagium vivum fluidum
Concept of virus as liquid continued until EM build in 1933 and showed first virus in 1939
What Exactly is a Virus — Are Viruses Living?
Not cells
They are essentially nucleic acid pieces wrapped in protein coats
Obligate parasites
Require a host to provide most tools, energy and macromolecules for their reproduction
All require host ribosomes for protein synthesis
They do not grow in broth media or use binary fission
Organism with two phases
Virion (infectious particle)
Virus is not living
Infected cell
Virus displays many properties of life
Viral Structure — Capsid
Roles
Attachment to host cell and entry into cell (spike proteins)
Protect the genome while between cells (nucleocapsid)
Use of one or few identically shaped proteins (capsomeres)
Viral Structure — Envelope
Not found on all viruses
More present in animal viruses > plant or bacterial
Consists of lipid bilayer
Attached to capsid by matrix proteins
Taken from cell membrane or other organelle as virus buds from a cell
Contains attachment proteins (removal of envelope will interfere with attachment)
Q: Which chemical disinfectants interfere with membranes?
A:
Viral Structure Impacts Tolerance to Chemicals
Naked viruses are almost always more tolerant of chemicals than enveloped viruses
Clumping (virus to virus or virus to cells or other organic matter)
Polio virus - alkylating agents
Foot & mouth disease - acids
Norwalk virus - chlorination
Viral Genome
May be either DNA or RNA, but never both
Double or single stranded regardless of nucleic acid type
Circular or linear
RNA viruses can be
+ strand (sense strand, same as mRNA)
- strand (antisense strand, complement to mRNA)
Q: Viruses have been found in Archaea. Given what you know of Archaea, which type of genome would be used?
A:
Baltimore Classification of Viruses
Based on genome type and how mRNA is produced
Viral Life Cycle — Attachment
Protein: protein interactions between viral receptor and cellular receptor
Some viruses may have co-receptors
Dictates tropism: can only infect cells that have cellular receptor
Introducing the proposed receptor into a cell that is not normally susceptible to infection will permit infection
Viral Life Cycle — Viral Entry / Penetration / Release of Genome
Naked and enveloped viruses may use receptor-mediated endocytosis (clathrin pathway) to enter cells
Binding of viral receptor to cellular receptor initiates the invagination of the cell membrane and coating of the vesicle with clathrin
Some viruses may escape vesicle prior to arrival in endosome while others rely on endosome for genome release
Enveloped viruses may use membrane fusion as a means to enter the cell. This may also be used as a means to escape the endosome
This process requires fusion proteins
Plant Viral Entry
Plant viruses often require damage to cell wall in order to penetrate the cell and then rely on mechanism described previously
Once inside they can quickly spread from cell to cell through plasmodesmata using movement proteins to shuttle genomic DNA or tubules to enlarge the plasmodesmata allowing viral capsids to pass
Phage Entry / Penetration
Bacteriophages rely on injection of their genetic material. The capsid typically stays outside the cell
Initial binding with tail fibers
Binding of tail plate (pins) possessing lysozyme activity
Injection by contractile motion
Viral Synthesis and Assembly
Synthesis varies considerably depending on the genome type
Some viruses use cellular polymerases, some bring their own
All viruses need the cell’s ribosomes for protein synthesis
Cells cannot carry out their normal functions since their molecular machinery has been hijacked by the virus
Viruses are capable of self-assembly
Formation of capsid from capsomeres and packaging of genome within the capsid
Rhinovirus is a member of the Picornavirus Family
The life of an RNA virus in a cell built for DNA
Our cells lack enzymes to synthesize RNA from RNA, which would be needed for transcription and replication of an RNA genome
Solutions
Bring your own RNA to RNA polymerase
Be a positive strand virus which can be translated immediately upon entry into the host cell
Translation of genome produces necessary enzymes for transcription and replication of genome
Polymerase synthesizes minus strand template which is used to make more + strand RNAs
+ strand RNAs are translated to make all necessary capsid proteins and simultaneously serve as genomes for new virus particles
General Characteristics of DNA Viruses
Can use the host cell transcription and replication machinery
Replication, transcription and translation proceed as normal
DNA synthesis in eukaryotes does not occur continuously but is confined to the S-phase of the cell cycle
Additionally, many differentiated cells rarely divide in multicellular organisms. Viruses must overcome this problem by inducing the cell cycle or replicating in rapidly diving cells
Q: What might happen if a virus interferes with the proper regulation of the cell cycle?
A:
Retroviruses
RNA viruses use the enzyme reverse transcriptase to create double stranded DNA from its RNA genome
This new DNA can insert directly into the host chromosome and function like a normal gene — being transcribed and translated, creating more viral particles
Risks Associated with Incorporation of Viral DNA Into Our Own
Insertion of viral DNA near oncogenes could alter their level of expression
insertion into an oncogene would disrupt its function
Either way will disrupt cell cycle and potentially lead to cancer
Viral Genome Type Can Impact Resistance to Chemicals
Viruses have a much higher mutation rate. RNA polymerases (RNA viruses) have an even greater mutation rate
High mutation rate + large population size = dramatic increase in chance of drug or chemical-resistant viral particle
Viral Release
Naked viruses often cause cell lysis - the loss of cells may result in pain or impair tissue function
Enveloped viruses are released by budding — possibly very slowly over long periods — chronic infections
Lytic vs Lysogenic ( latent, temperate )
Viruses can just hang out in a cell by incorporating its genome into that of the cell
Every time the cell replicates, the virus is replicated with it
Good strategy if cells are happy and growing
Lytic Process
Phage attaches to host cell and injects DNA
Phage DNA circularizes and enter lytic or lysogenic cycle
New phage DNA and proteins are synthesized ans assembled into virions
Cell lyses, releasing phage virions
Lysogenic Process
Phage attaches to host cell and injects DNA
Phage DNA circularizes and enters lytic cycle or lysogenic cycle
Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage
Lysogenic bacterium reproduces normally (many cell divisions)
Occassionally, the prophage may excise from the bacterial chromosome by another recombinaton event, initiating a lytic cycle
Quantifying Viral Numbers - Titer
Plaque assay: agar restricts the diffusion of virus so it simply moves to surrounding cells forming a plaque as cells die
For viruses that do not cause cell death they can engineer the virus with a gene that produces color inside cell
For lytic virus, not budding virus
ELISA: enzyme linked immunosorbent assay
Detects the presence of virus
Amount of virus is proportional to amount of color
Detection and Quantification of Viral Numbers
PCR product is not always equivalent to infectious virus
Phage Therapy
Originally proposed and tried in early 1900s
Increase in antibiotic resistance has sparked renewed interest
FDA approved phage treatment as preventative measure on lunch meats (against listeria)
Several researchers / biotech companies are designing phages against specific pathogens
Several trials done in mice; clinical trials in humans are underway
Give orally, rectally, pills, liquids creams, injections, tampons
New COVID Drug
Molnupiravir
A new drug to treat COVID is proposed for release
Based on the chemical structure, how might it target the virus?
Q: You are drawing blood from a virally infected patient and accidentally poked yourself with a needle. You are told to start an antiviral medication to prevent possible infection. What type of med (target) might you use for this?
A:
CRISPIR / CAS System
When bacteria are infected by a virus, they may use their CRISPR system to cut up the invading viral DNA and insert pieces of it (spacers) into their own genome as a “memory” of the infection
Using this system, bacteria can collect sequences from many different infecting viruses to create a library. Since the CRISPR sequence is contained in genomic DNA, it is passed on to each generation, and the library continues to change ad adapt to more common threats over time
Bacteria transcribe the spacers into RNA, which can form a complex with the Cas9 enzyme
These complexes monitor the cell for any DNA sequence complementary to the RNA
If matching (viral) DNA is encountered, the spacer RNA-Cas9 complex binds to it and cuts the viral DNA to prevent it from replicating. This halts the viral infection
Cas9 enzyme (Cas9): an endonuclease that cuts both strand of DNA at a specific site
Single guide RNA (sgRNA): has guiding region - is complementary to the target that defines the DNA sequence that Cas9 cuts
Scaffold region: forms hairpin loop structure (scaffold) that binds in a crevice of the Cas9 protein
Protospacer adjacent motif (PAM): this sequence motif is immediately downstream of the target sequence. Cas9 recognizes the PAM sequence 5’-NGG
If you know your target sequence, you can design a guide RNA to cut at a specific site
What sequence would be used to cut the DNA at the red dotted line?
What happens after the cut?
Insert new gene or repair broken gene
Knock out gene function
Coronary Artery Disease (CAD)
Lowering levels of low density lipoprotein (LDL) cholesterol has been shown to effectively reduce risk of CAD
LDL receptors (LDLR) in the liver clear LDL from blood plasma
Levels of LDLR are themselves reduced by proprotein convertase subtilisin / kexin type 9 (PCSK9), a serine protease that binds and degrades the receptors
Reducing PCSK9 would increase LDL receptors and therefore lower LDL to reduce risk of CAD
A goal of gene-editing therapy may be to reduce or eliminate PCSK9 enzyme function.One strategy is to disrupt the gene by making a cut within exon 1 and allowing nonhomologous end joining (NHEJ) to occur. This strategy would reduce levels of functional PCSK9 enzyme in the liver, which would reduce degradation of the LDL receptors to allow more removal of LDL cholesterol from the bloodstream.
How would we design a proper guide RNA to target this sequence?