numbe r 5 Done by Mustafa Khader Corrected by Mahdi Sharawi Doctor Ashraf Khasawneh
Viral Replication Mechanisms: (Protein Synthesis) 1. Monocistronic Method: All human cells practice the monocistronic method of replication, which means that each gene codes for only one protein. This is of importance in viruses as segmented viruses also use this method, where again each segmented gene codes for one mrna which in turn codes for one protein. 2. Formation of polyprotein: We have previously seen viruses such as the Human Immunodeficiency Virus (HIV) that uses a replication strategy involving the translation of a large polyprotein, which is then cleaved by viral proteases into smaller individual proteins. Positive sense RNA viruses are capable of using this method of replication and protein synthesis. 3. Make a primary transcript and use alternative splicing (Dr. Ashraf did not really emphasize on this point) 4. Include special features in the mrna which enable ribosomes to bind internally: In this method the ribosome detects and binds internally to the mrna allowing the detection of where the gene begins and where it ends, synthesizing a single protein only. This figure summarizes the replication strategies seen in all viruses except the Hepatitis B virus Assembly:
We now have all the components required for the assembly of new viruses which are the viral proteins (both structural and functional) and the newly formed viral genome. Assembly involves the collection of these components required in a certain site within the host cell. This site is determined by the site of replication of the virus within the cell, and the method/mechanism of release of this particular virus. The mechanism and site of release of enveloped viruses are determined by the M 2 protein of the virus. This protein will arrange at a certain point in the cell membrane dictating the viral point of release. Also in the case of enveloped viruses further components needed such as spikes or glycoproteins are assembled outside of the host cell, and the envelope is acquired from the host cell s cell membrane. Thus, for example picornaviruses, poxviruses, and reoviruses assembly occurs in the cytoplasm while on the other hand, adenoviruses, polyomaviruses and parvoviruses assembly occurs in the nucleus. Each specific capsid structure undergoes a different method of assembly: 1. Helical: The capsomeres form rings, these rings are stacked on top of each other and the genetic material winds up in the hollow spaces of these rings. 2. Icosahedral: The capsomeres are arranged in the Icosahedral structure leaving a cap at the top that is left open, the genetic material will enter the capsid through this cap forming our nucleocapsid. Finally, formation of the nucleocapsid finishes with closure of the cap. Maturation: This is the stage of viral-replication where the virus becomes infectious. During this stage there are usually structural changes to the viral particle most commonly induced by specific cleavages of the capsid proteins causing conformational changes in the viral proteins. The enzymes used in this stage can be viral proteases (most common), cellular enzymes, or in some instances a mixture of both viral and cellular enzymes can also be used. Some viruses mature within the cell, while others mature after release. It all depends when the cleavage of the proteins occurs, and the virus reaches its final conformation. Release:
All viruses (apart from plant viruses which need to overcome the plant cell wall) are released by either one of the two following mechanisms: 1. Lytic Process: The mechanism by which most naked viruses are released, it is fairly simple, the cell breaks open (is lysed) causing the release of all the virus particles within it. 2. Budding: This is the mechanism by which enveloped viruses are released from the host cell. As stated previously the point at which budding occurs is dictated by the point of binding of the viral M 2 protein to the cell membrane of the cell. Once again, the envelope is acquired from the cell membrane of the cell. Viral Genetics As we have seen in previous lectures viral genome can either be DNA or RNA, where RNA viruses also have the option of being either segmented (ex. H. Influenza and Rotavirus) or non-segmented genomes. Viruses grow rapidly producing many viruses after their infection of a host cell, Which means that a vast number of genetic replications occur in a small amount of time. This in turn gives DNA viruses an advantage over RNA viruses as DNA viruses have the ability to proofread (scan the DNA strand for any mistakes and fix these mistakes if found) their DNA following its replication. In turn this causes the occurrence of mutations in DNA viruses significantly less than in RNA viruses. This is due to the fact that RNA viruses encode their own enzymes (RNAdependent RNA polymerase and in certain cases Reverse Transcriptase) and unfortunately these enzymes lack proofreading, rendering then unable to correct any mistakes that may arise during the process of replication. Every 8-10 thousand nucleotides added by these enzymes there is usually a mistake causing a mutation to the viral genome. There are two changes that may occur to the viral genome: 1. Mutations: are usually point mutations, where a single nucleotide base is changed. a. Origin of mutations: i. Spontaneous: Due to the reason stated previously ii. Physically induced: UV light and X-rays for example iii. Chemically induced: Hydroxylamine (NH 2 OH) or Alkylating agents
b. Types of Mutations: i. Point Mutations (three types) 1. Silent: In this type of point mutation the replacement occurs at the third base in the codon base thus usually/most probably encoding the same amino acid. This is due to the fact that most amino acids are encoded by four codons (differ in the third base only). This isn t always the case as there are amino acids that are coded by two codons only, thus a change in the third base may give rise to a different type of mutation. This specific type of mutation (silent mutation) has no effect on the functioning of the protein as the newly formed codon specifies the same amino acid, resulting in an unmutated protein. 2. Missense: The replacement occurs at the first or second base of the codon thus encoding a different amino acid. This may have drastic changes to the shape or function of the protein (the change we see in sickle cell anemia) or amazingly very minimal or no visible change to the protein 3. Nonsense: A change in a base in the codon gives rise to a stop codon causing the premature termination of translation causing the formation of an unusually short protein, therefore, most of the time this mutation gives us a non-functional protein. ii. Insertion or Deletion: A nucleotide base is added or removed respectively, causing frame shift, which in turn causes all the amino acids subsequent to the insertion or deletion to change forming a completely non-functioning protein. (Insertion causes frame shift to the right, while deletion causes frame shift to the left) 2. Recombination: is another form of genetic variability between viruses, it is described as the exchange of information between two genomes. We have three types: a. Classical Recombination: Most commonly found in DNA viruses, where a certain segment is exchanged between two genomes causing exchange of a sequence of nucleotides between the two.
b. Copy Choice Recombination: Found in RNA viruses, in this form of recombination the RNA-dependent RNA Polymerase and Reverse Transcriptase enzymes have the ability to jump from one template to another. Thus, the enzyme will start forming a complementary strand to the first template strand until it reaches what is so-called a stop sign. The enzymes along with the complement strand currently being formed then jumps to the second template strand and continue the process of replication. Therefore, the newly formed strand has genetic characteristics acquired from both the first and second strands. The length of the newly formed genome depends on the site on the second template where the enzyme jumped to thus the encoded strand may be longer, of the same length, or shorter than the original strand. c. Re-assortment: In segmented viruses, we have what is called re-assortment/ genetic shift. For example, in the Influenza virus we have what is known as Antigenic shift or we can have Antigenic drift.
i. Antigenic drift occurs as a consequence to a point mutation as a consequence to the lack of proofreading in RNA-dependent RNA Polymerase every 8-10 thousand nucleotide bases. This mutation can occur in any of the segments of the influenza virus which is of uttermost importance if the mutation occurs in the spikes/glycoproteins. The spikes/glycoproteins are considered to be the most antigenic part of the virus due to the fact that they are in direct contact with the immune system, and antibody attachment to the virus is mediated by these spikes/glycoproteins. In the case of the influenza virus there are two types of spikes, known as HA (hemagglutinin) and NA (neuraminidase). It has been discovered that the antibodies synthesized are complementary to hemagglutinin. Thus, if a point mutation occurs in hemagglutinin the efficacy of antibody binding may decrease due to minor changes to the shape of hemagglutinin due to the point mutation. Every approximately 3-5 years a more significant change occurs due to several point mutations affecting structural amino acids in an important site in the glycoprotein. This induces a change to the overall shape of the glycoprotein which decreases the efficacy drastically causing it more difficult to neutralize the viruses. ii. Antigenic Shift/Re-assortment: More than one species of a certain virus for example: Human influenza virus, Bovine influenza virus (from birds), and Swine influenza virus (from pigs) infect a single cell. When this occurs, each species is going to replicate its viral genome and the problem occurs at the stage of assembly. Each Influenza virus has 8 segments, if there is a mixture of all three viral genomes in one virus following assembly then traits that were not previously found in the virus will arise. The most severe problem will occur if the gene that encodes for hemagglutinin is acquired from another species, causing our antibodies to be completely unable to bind to the virus. This in turn causes the new virus to be extremely infectious, elicit very severe infections, and cause a high mortality rate. This antigenic shift/ re-assortment occurs every 7-10 years. It is a very efficient form of non-classical recombination that can occur only in segmented viruses only (can occur naturally), and is being used in the synthesis of newer vaccines for both the Influenza and Rota viruses.
If we want to rate the efficiency of viral replication we can soundly conclude that viral replication is a relatively inefficient process as many of the newly formed virus particle are usually viruses that lack the ability to infect other host cells. This is because many mistakes can occur during the process of viral replication, these mistakes account for causing approximately 60% of the new viruses to be non-pathogenic, while only 40% of these new viruses to have infectious properties. All the previous methods of genetic exchange (classical recombination, copy choice, re-assortment) do not occur in non-segmented negative strand RNA viruses, and these viruses have the least ability to exchange their genetic material. Vaccines We have a Rotavirus vaccine (uses the previous principle) in which we look for the most immunogenic part of the Rotaviruses (the surface proteins) of the previous year and we add them to an attenuated virus. An attenuated virus is a live virus that has been modified to have reduced virulence. We then allow the virus to replicate in vitro and we administer it to children via oral drops. This vaccine is given specifically as oral drops due to the fact that the original virus infects the host through the oral-fecal route thus administering the vaccine orally will allow the attenuated virus to mimic the infection process of a regular Rotavirus. The main idea behind this is to expose the immune system to the virus enabling it to form antibodies and B-memory cells for this virus. Therefore, in the case of a subsequent infection with the actual virus the immune response will be very rapid and efficient, and the virus will be neutralized. In the case of the Influenza virus we have two types of Influenza Vaccines: 1. Live attenuated virus: The virus becomes attenuated by allowing its growth at suboptimal temperatures, as the virus is injected into eggs and placed in an incubator and they lower the temperature from 33/34 o (optimum temperature) to 24 o causing several mutations to occur to the virus causing it to have lower virulence but still have the ability to replicate. The most antigenic parts HA (hemagglutinin) and NA (neuraminidase) which are acquired from the most infectious viruses in the previous year/s are added to this attenuated virus. The most infectious viruses are determined by the number of people they infected, and which virus was associated with the highest mortality rate. For
example, we put two Influenza A and one Influenza B in the vaccine determined by how infectious they were in the past year or couple of years. This vaccine is modified annually as each year the most infectious virus may change. This vaccine is once again administered as a live attenuated vaccine intra-nasally, entering the upper respiratory tract and mimicking the infection of the original influenza virus eliciting an immune response. 2. Shot vaccine: You can, for instance, grow two strains of Influenza A and one strain of Influenza B viruses separately in eggs and allow them to replicate. Then you harvest them and kill them using Formalin. Thus, we have now acquired a dead virus that has the most antigenic structure (HA) still present. We can then administer this vaccine by route of injection allowing the immune system to recognize the antibody without the risk of infection. Note: Live attenuated vaccines are contraindicated in immunocompromised patients or patients who are taking immunosuppressants due to the fact that anything that is weak will try to make itself stronger, same goes for the virus. The attenuated virus will start trying to make itself stronger, and with its return to its optimal temperature aiding in this process, it will most likely be successful. Thus, a person that is immunocompromised will require a longer duration of time to form antibodies and memory T-cells and during this time we are allowing the virus to get back to the wild (diseased) typecausing a highly un-wanted infection to the person at hand. Defective and Helper Viruses As we have stated before most viral replication is considered to be inefficient due to the numerous number of mutations that may arise. These mutations provide us with defective viruses, which are viruses that have inefficient genetic material and lack the ability to replicate and form infectious progeny viruses. On the other hand, we also have what are called helper viruses, which supplement the genetic deficiency and allow the defective virus to replicate and form infectious progeny viruses. This occurs when both the defective and helper viruses infect a host cell simultaneously. For example, let s imagine that the defective virus lacks a certain enzyme. When they both infect a cell, the defective virus will compete with the helper virus for the resources and when assembly occurs the defective virus might take the missing enzyme from the
helper virus, becoming infectious again. So, during assembly the defective virus will take the missing component from the helper virus to re-instate its infectivity. Therefore, if the virus was infected with the defective virus only then there would be no actual infection and all the viruses would die within the cell as they lack an infectious property and no replication will occur. On the other hand, if the efficient virus infects the cell then after replication, formation of infective and defective viruses will occur. However, if both of these viruses infect a certain cell together then they will complement each other, but this does not necessarily mean that the virus will become infective. Von Magnus Phenomenon: A scientist (Von Magnus) found that infecting cells with Influenza Virus at high MOI (Multiplicity of Infection) was associated with decreased number of infective viruses, while infecting cells at low MOI was associated with increased number of infective viruses (inverse relationship). This is because, if you have a lower number of viruses the chance that a single cell will get infected by more than one virus is low, therefore if this cell is infected by a defective virus then this cell will not produce any infective viruses. Only cells that are infected with infectious viruses will produce viruses that are capable of infecting other cells. We also acknowledged before that about 50% of the viruses produced are effective and the other 50% are defective. So, we end up with a significant number of infective viruses. On the other hand, if we infect 1000 cells with 10000 viruses (high MOI) then the probability that a cell will be infected with more than one virus is higher. These viruses can either be infective or defective so if an infective and defective virus infects one cell they will once again complement each other. We would expect this to cause more infectious viruses to arise. Contrary to our expectations, the viral genome in the non-infectious/defective virus simply cannot become infectious even if the missing enzyme was acquired during assembly. Thus, 70% of the newly formed viruses will be non-infectious, while only 30% will be infectious. This might be a bit confusing, but it is fairly simple, the virus lacks the infectious genome, so even if it were to acquire the missing component the genome is not
viable to infect other cells. Therefore, complementation with a helper virus is only at the functional level (proteins and enzymes) and has no effect at the level of nucleic acids.