4.9 - Anti-viral agents Flashcards
1
Q
What is a virus?
A
- viruses are infectious obligate intracellular parasites
- reliant on host machinery
- genome comprises DNA or RNA
- within an appropriate cell, the viral genome is replicated and directs the synthesis, by cellular systems, of more viral components and genomes
- the components affect the transport of replicated viral genomes through the environment to new host cells
- viruses seen through electron micrographs of negatively stained viruses
- viruses are small - 10nm to 1um
2
Q
Non-enveloped and enveloped viruses
A
- non-enveloped viruses have a symmetrical protein capsid as their outside layer e.g. adenovirus, picornavirus, calicivirus
- enveloped viruses have a lipid envelope derived from host membrane as an outside layer - can be pleiomorphic (e.g. measles virus) or typical shape (e.g. Ebola virus)
- some viruses have a combination of capsid (tegument) and envelope like herpes
3
Q
What is the central dogma of molecular biology?
A
- DNA is transcribed into RNA, and RNA is translated into proteins
- significant for viruses as some have an RNA genome but use reverse transcriptase to convert it into DNA in cell
- some viruses carry RNA in negative sense - complementary strand of mRNA –> must convert negative sense back into complementary copy (positive sense) which is then translated by ribosome, in order to translate genome into a protein
4
Q
What are the consequences of the viral genome type?
A
- RNA viruses and retroviruses use their own polymerase to replicate, which lack proof reading capacity leading to high mutation rate
- RNA viral genome are limited in size due to inherent instability of RNA vs DNA –> largest RNA viruses are coronaviruses –> RNA viruses often use complex coding strategies to make more proteins than expected from a small RNA genome e.g. overlapping reading frames = different proteins
- DNA viruses have genomes up to 100s kb = plenty of room for accessory genes that can modify the host immune response –> these genes often lost in passage in culture
- segmented genomes (physically discrete sections of nucleic acid encode different genes) allow an additional easy form of recombination called reassortment, but also impose more difficult packing strategies –> influenza has 8 different RNA segments, rotavirus has 11
5
Q
Generic virus replication cycle
A
- virus has protein in its capsid/envelope that attaches onto a specific virus receptor on host cell membrane
- capsid falls away –> nucleocapsid remains, exposing viral genome
- genome replicated –> mRNA –> proteins –> assembly
- host cell tends to die as pathways recognise that the cell is infected (but virus can still spread to other cells)
6
Q
HIV replication cycle
A
- fusion of HIV to the host cell surface - engages with receptor
- virus and host cell fuse and contents released into cell - HIV RNA, reverse transcriptase, integrase and other viral proteins enter the host cell
- viral DNA formed by reverse transcription
- viral DNA transported across the nucleus and integrates into host DNA using integrase
- new viral RNA is used as genomic RNA and to make viral proteins
- new viral RNA and proteins move to the cell surface and a new, immature HIV forms
- virus matures by protease, releasing individual HIV proteins and virus budded out of cell
7
Q
Influenza replication cycle
A
- flu virus attaches via glycoproteins and glycolipids on surface
- cell takes in viral particle by endocytosis
- virion fuses with endosome lipids to release the 8 RNA negative sense segments
- they enter the nucleus where RNA-dependent RNA polymerase copies them into mRNA and replicates them into new genomes
8
Q
How do we investigate viruses in the laboratory?
A
- cytopathic effect (death of cell caused by virus) - usually a result of the virus lysing the cell
- could be due to the shut down of host protein synthesis or accumulation of viral proteins
- viruses form plaques in cell monolayers - virus kills a bunch of cells in the middle of the layer
- syncytia - viruses with surface proteins that can fuse at neutral pH often fuse cells together - measure of syncytia is also a measure of how many viral particles were there
9
Q
What are the different ways we can detect/diagnose viruses?
A
- detecting viral genome - PCR, RT-PCR
- detecting viral antigen - IFA, ELISA
- detecting virus particles - EM, HA
- detecting virus cytopathic effect in cultured cells (virus isolation)
- detecting antibodies to virus (serology) - useful for counting how many people infected during outbreak
10
Q
How can viruses be manipulated?
A
- virus genomes are so small they can be synthesised
- when introduced into permissive cells, the cells think they have been infected by the virus and are driven to synthesise components of new viruses which are made de novo
- this allows reverse genetics - the creation of viruses at will with engineered mutations in their genomes
11
Q
Why are viruses so difficult to control?
A
- since they are intracellular parasites, a lot of how they work is completely dependent on host cell machinery which we cannot target, or we will harm the infected person
- this means it is hard to achieve a therapeutic index - ratio between how much drug you have to use in order to control the virus : the amount that makes a person feel ill from the side effects of the drug
- we need selectivity and specificity to find processes the virus does which the host cell does not
- only a few effective antivirals exist (whereas there are many effective antibiotics, as bacteria function very differently to human cells)
- e.g. in bacteria, ribosomes and cell walls are different, but viruses use our own ribosomes and lipid cell membranes
12
Q
What are targets for antiviral drugs?
A
- viral enzymes –> increased understanding of viral structure and components can lead to rational drug design
- others act as nucleoside analogues to inhibit / interfere with nucleic acid replication, but need to achieve some element of specificity for the viral polymerase (so our own DNA is not affected)
- some drugs target specific viral factors - directly acting antivirals - usually specific for a particular virus and so their use must be coupled with appropriate diagnostics
13
Q
What is acyclovir?
A
- best example of an antiviral agent with specificity
- nucleoside analogue that looks like guanosine but the bottom half of the molecule is missing, so 3’ hydroxy group is not there for other nucleosides to attach to –> it is a chain terminator
- lack of 3’ hydroxy group prevents phosphodiester bond formation which is essential for growing the chain
14
Q
Where does acyclovir’s specificity come from?
A
- it is only activated inside virus infected cells
- given to patients as a pro-drug in unphosphorylated form - all nucleosides need to be triphosphorylated to be used in DNA/RNA chains
- specificity largely due to phosphorylation of acyclovir (ACV) to acyclovir monophosphate (ACVMP) by virus-encoded thymidine kinase
- human thymidine kinases cannot do this first phosphorylation so we can take as much ACV as we want and it will not harm healthy cells
- subsequent phosphorylation to acyclovir triphosphate (ACVTP) by cellular enzymes
- ACVTP has higher affinity for viral DNA polymerase than for host cell polymerase
- resistance to ACV is rare but would most likely be caused by mutation in thymidine kinase in a way it can no longer phosphorylate ACV
15
Q
What is remdesivir?
A
- analogue of adenosine, causes chain termination 3 nucleotides downstream of incorporation as it twists the shape of the growing DNA/RNA molecule so new nucleotides cannot be added downstream
- developed against hepatitis C
- tested against Ebola but didn’t meet endpoint
