Viruses genomes are made from RNA or DNA encased within a protein shell. They rely on the host cell for all their reproductive functionality and consequently are difficult to target. They have very few unique components and so selective drugs against viruses are hard to come by, and are often toxic to the host cell as well. They also mutate very quickly and are hard to study because they cannot be grown in culture, instead needing to be assembled by cells.
When a virus infects an animal cell, the cell will start producing interferons as a response. INF alpha and INF beta can block viral replication. Double stranded RNA which is a common symptom of viral infections is a trigger for the release of INF alpha and beta. These interferons bind to interferon receptors in both infected and neighbouring cells. This activates a phosphorelay signal which signals certain antiviral genes to be expressed. NK cells which are a class of immune cells are also activated by interferon and will destroy virus infected cells.
Moreover some proteins such as oligoadenylate synthetase convert ATP into 2’-5’ linked poly(A) meaning that the virus is starved of an ATP source. 2’-5’-poly(A) activates RNA endonucleases to break down viral genomes. It also activates P1 kinase which halts protein synthesis.
Mx proteins are another class of proteins (GTPases) which form a ring around viral RNA, preventing RNA polymerase from replicating the viral genome. It is the difference in animal Mx proteins, and the susceptability of different viruses to different Mx proteins which govern how well a virus can jump from one species to another.
RNAi as an antiviral treatment
RNAi is the natural cell defence mechanism against double stranded RNA. Some viruses have protection mechanisms against RNAi, but injecting short interfering RNAs into mammalian cells triggers a response that even these viruses cannot withstand. These siRNAs are designed to be the same as highly conserved pieces of the viral genome. siRNAs can be taken into the body by inhalation and are effective against influenza, parainfluenza and measles. RNAi can also be used to confer viral resistance upon plants to increase crop yields. This is done by cloning in DNA which when transcribed folds into short hairpin RNA, this triggers RNAi and the plant becomes resistant to that virus.
Influenza is a (-)ssRNA virus. Its genome is segmented into 8 pieces of ssRNA, with each piece being about 890-2341 bp long. The pieces are encased in a nucleocapsid with an RNA polymerase attached to the end ready to start replication of the genome. The outer membrane surrounding this has viral proteins called neuraminidases and hemmaglutinins and various ion channels. These H and N proteins differ from strain to strain.
Viruses are engulfed into cells inside a vesicle. They bind to the cell surface cognate receptors and the virus is taken in by vesicle mediated transport. The vesicle and outer coat of the virus dissolve and the nucleocapsids enter the nucleus, disassemble and release the naked RNA. Here the virus replicase turns the (-)RNA into (+)RNA. The RNA is replicated and then leaves the nucleus as mRNA, travelling to the cytoplasm. This mRNA codes for proteins for virus assembly. Recall that the influenza genome is segmented into 8 pieces of RNA. If two different influenza viruses infect a cell at the same time, then during viral assembly these DNA pieces may be exchanged between viruses, causing a new virus to be formed which has bits of DNA from both viruses. This allows for high genetic diversity of the flu, and is one of the reasons why a new strain emerges every year or two.
Influenza B is mostly a human influenza with not much genetic variation but influenza A infects “people, pigs and poultry”. Different hosts lead to more genetic diversity due to shuffling of different viral genomes in different hosts meaning that less common but more severe epidemics are caused by it.
Amantadine is a the first flu therapeutic drug. It binds to the M2 protein which is found on the outer membrane of influenza A. This blocks the function of the ion channel meaning the virus cannot uncoat and so the virus genome remains locked within the particle. This molecule is not effective against influenza B because it does not have the M2 ion channel. Due to the nature of its activity, it has to be given early to be effective.
Neuraminidase inhibitors are another class of antivirals. One such example is Tamiflu. Neuraminidases usually cleave N-acetylneuraminic acid, but the drug is an analog of this acid and blocks their action. If this happens then the virus becomes trapped inside the infected cell, however mutations in the N proteins is leading to resistance to these drugs.
HIV is a virus which infects T cells, which are white blood cells. It uses the native CD4 receptor on the surface of these cells, its gp120 protein on its outer coat recognises the CD4 receptor and binds to it which allows the virus to enter. The damage caused to the T cells by HIV cripples the immune system function leading to possible death by opportunistic infections. HIV DNA integrates into the DNA of the T cell and the genes begin to be expressed, making more virus particles. The gp120 virus envelope protein inserts itself on the T-cell membrane, causing T-cells to clump together as the CD4 receptors bind to one another. The cells fuse and then die. Because 70% of the T cells have the CD4 receptor, the death of these cells leads to the immune system fading away over 10 or so years.
Chemokine receptors are co-receptors for HIV that bind chemokines. CCR5 is important in the entry of HIV into T cells. Some mutations in CCR5 lead to natural HIV immunity, which about 2% of Europeans are homozygous for.
50% of antiviral drugs have been developed specifically to target AIDS. The problem is the high mutation rate of HIV because it is an RNA virus. The mutation rate is 1 base per genome per replication cycle. In a single patient HIV exists as many different quasi-species. This is dealt with by clobbering the virus with different drugs all of which target different aspects of the virus.
AZT is a a DNA chain terminator. It is essentially a thymine without the 3’-OH end meaning that when it gets incorporated into the growing DNA of a viral genome during replication, it has to stop as the polymerase cannot find a 3’ end. This drug has the drawback that it can affect uninfected cells too. Acyclovir is another example of a drug working in a similar way using guanine instead of thymine.
HIV proteins are made as polyproteins which are then cut into individual proteins by the HIV protease. Inhibiting polyprotein cleavage prevents assembly of the virus particle and therefore protease inhibitors are drugs that do this. These can be amino acid analogues which bind non-productively to the protease, inactivating it.
The prion protein (PrP) exists either as normal protein in the cellular form: PrPc, or as pathogenic protein: PrPSc. PrPSc binds to PrP and causes it to misfold itself, becoming PrPSc, so a small amount of PrPSc can overwhelm all the PrP. This leads to neurodegenerative diseases which worsen over time. Proteins wich a higher chance to misfold come from mutations in the Prnp gene. Normal proteins can misfold at a rate of 1 in 1 million people causing a spontaneous prion disease. These misfolded proteins aggregate into amyloids, which are fibrils with high beta-sheet content. These can be transmitted to other people, and so prion diseases can be infectious.
Well known prion diseases are scrapie (in goats and sheep), Kuru, mad cow disease and chronic wasting disease (in deer and elk).
Detecting prion diseases
At first, because there were no antibodies to differentiate between PrPc and PrPSc, and because PrPSc is very resistant to proteases, the first assays used protease to degrade all the PrPc and then identify prions using western blot. New isoform specific antibodies are used in conformation-dependent immunoassays (CDIs). These have been coupled with amplification in order to detect tiny amounts of infectious prion. To do this, PrPSc is mixed with surplus PrPc. PrPSc conerts PrPc to PrPSc, causing aggregates to grow. The sample then gets sonicated to break up aggregates into smaller pieces to begin the cycle again. One can expect a 60-fold increase in 5 cycles. This is called Protein Misfolding Cyclic Amplification (PMCA) and can be used to detect sub-femtogram levels of misfolded protein in the starting sample.
References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.