LESSON 4.4 WORKBOOK. How viruses make us sick: Viral Replication
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1 DEFINITIONS OF TERMS Eukaryotic: Non-bacterial cell type (bacteria are prokaryotes).. LESSON 4.4 WORKBOOK How viruses make us sick: Viral Replication This lesson extends the principles we learned in Unit 4.1 by addressing how viruses cause host damage. It focuses on how viruses replicate and then exit from host cells. As with intracellular bacteria, viruses may damage the host cell either directly or indirectly. In addition, we will focus on how mutations and adaptations that arise during viral replication contribute to pathogenicity and ultimately host damage. Viruses are designed to make more virus. Before we begin to discuss viral replication we need to briefly review the main processes of the viral life cycle. As the picture above illustrates, viruses have receptors on their surface used to attach to and enter target host cells. Viruses then use the cell s own machinery to to replicate their genomes (which, unlike eukaryotic cells may be in the form of RNA or DNA) and to make the proteins they need. The virus then assembles with the help of the host cell. Once the viruses are mature, they exit to spread to a new host cell by budding off of or lysing the cell. Review: What receptors do H1N1 and HIV attach to on host cells? Review: How do H1N1 and HIV exit host cells (are they naked or enveloped)? Lesson 4.4 1
2 LESSON MATERIALS HIV How do viruses cause damage to the host? Like intracellular bacteria, viruses replicate in host cells. Therefore a virus can directly damage host cells by depleting their nutrients and by causing the cell to lyse upon exiting the host cell. In addition, viruses can indirectly damage host cells when infected cells are killed by T cells. This may happen when an infected cell is killed intentionally or when bystander cells become casualties. For example, H1N1 infected epithelial cells in the lungs will be targeted by T cells, however, neighboring epithelial cells may also be damaged in the process. As always, when cellular damage is extensive, the functions of the tissues can become compromised, which leads to disease. During viral replication the viral genome is duplicated multiple times, viral proteins are made, and the virus is assembled. This process can compromise the infected cell because the cell s resources are diverted to make virus. The next few pages focus on the processes involved in viral replication after the viral genome is in the host cell cytoplasm. Viral replication is a critical step in the viral life cycle that uses host cell nutrients. During viral replication, the viral genome is duplicated multiple times, viral proteins are made, and the virus is assembled. This process can compromise the infected cell because the cell s resources are diverted to make virus. As we will see, the processes of replication depends on the type of genome that a virus has; Viruses can have genomes that are made of DNA, like cells, or RNA. How do viruses cause direct and indirect damage to the host? Lesson 4.4 2
3 Influenza virus DEFINITIONS OF TERMS: LESSON MATERIALS How viruses use and misuse molecular dogma Molecular dogma explains the process of DNA replication, RNA transcription and protein synthesis (translation). To understand how different types of viruses manipulate the cells resources, it will be helpful to do a quick review of how molecular dogma works. The diagram to the right shows the three stages of molecular dogma as you learned it in Bio one. DNA replication: One strand of DNA is copied to make an identical second strand of DNA. Transcription: The DNA is used to make a strand of RNA. The sequence of the RNA strand is complementary to the DNA sequence. Translation: The sequence of the RNA is used to make the corresponding chains of amino acids that then fold into the functional proteins the cell needs to survive. The virus needs to be in the right part of the cell at each stage. Remember that DNA replication, transcription, and translation take place in defined locations inside the cell. Replication and transcription happen in the nucleus, because this is where the DNA and enzymes needed in transcription are. Translation takes place in the cytoplasm, again because this is where the proteins for needed for this process reside. This means that RNA needs to be transported from the nucleus to the cytoplasm after transcription, so that it can be used for translation. Hence, viruses need to be in the right part of the cell to exploit molecular dogma. For example, if a virus needs to use the host replication or transcription proteins the virus needs to get to the nucleus. Furthermore, to make viral proteins the viral RNA needs to be in the cytoplasm. What host proteins will a DNA virus need to replicate its: Viral proteins (capsid etc)? Genome? Lesson 4.4 3
4 LESSON MATERIALS Viral replication uses and manipulates normal cellular processes Since DNA viruses have a DNA genome, they need to be in the nucleus to access the replication and transcription apparatus of the cell. In fact, all viruses that use DNA need to enter the host nucleus. Just like host genes, the viral genome follows molecular dogma and uses host enzymes: 1. DNA polymerase: performs DNA replication. 2. RNA polymerase: performs transcription. 3. Ribosomes: perform translation. But if a virus needs to do something that a host cell cannot do, the virus must enter the host cell with all the necessary proteins to be able to do it for itself. For example: Host cells never replicate RNA, so how does a virus with an RNA genome replicate itself? the answer is that the virus needs to bring all the machinery it needs to replicate RNA along with it. Some viruses defy molecular dogma by making RNA from RNA. This diagram illustrates how the RNA virus manages to replicate its genome in a host cell that can only replicate DNA. The viral enzyme that makes RNA from the virus genome RNA is called RNA dependant RNA polymerase. The virus brings this protein with it when it infects the cell. RNA-dependent RNA polymerase is unique to each virus, and is not found in healthy host cells, so it may be a Where would each of the enzymes involved in the processes of molecualar dogma be located? Lesson 4.4 good drug target! 4
5 DEFINITIONS OF TERMS: Codon: a set of three nucleic acid base pairs (DNAs building blocks) that code for one amino acid. LESSON MATERIALS Retroviruses defy molecular dogma by making DNA from RNA and then inserting their DNA into the host s genome. Retroviruses are more advanced than most DNA and RNA viruses because they can permanently insert (integrate) their genome into the genome of the host cell. Retroviruses, such as HIV, have an RNA genome that they then make into DNA with a special enzyme called reverse transcriptase. The retroviruses bring the gene for reverse trasncriptase with them when they infect the host cell. The first thing they do is to make a DNA copy of their genome using the reverse transcriptase. Then, another viral enzyme, integrase, inserts the viral DNA into the host s genome. The retrovirus also brings the gene for integrase with it when it infects the host cell. Once the viral DNA has been integrated into the host cell genome it is copied using regular molecular dogma whenever the host cell tells it to. Reverse transcriptase and integrase are viral proteins that are unique to each virus, so just like RNA dependant RNA polymerase they may serve as drug targets. RNA dependant polymerase and reverse transcriptase cannot proofread, so the copies of the viral genomes are filled with errors. When DNA and RNA is replicated, random errors arise at a rate of about 1 in 100,000 base pairs. However, codons with different nucleotide sequences can code for the same amino acid, so some errors will not impact the eventual protein (this is called a silent mutation). In contrast, mutations that change the amino acid sequence and structure of the protein could affect pathogenesis. These mutations are the foundation for new adaptations, and this process of random mutation is called antigenic drift. Why would reverse transcriptase make a good drug target? Lesson 4.4 5
6 DEFINITIONS OF TERMS: Virulence: the degree of infectiousness of a pathogen. LESSON MATERIALS DNA Proofreading. Mutation at a rate of 1 in 100,000 is far too high for eukaryotic cells to tolerate. If our cells had mutation rates like this, a cell would rapidly become defective and maybe even cancerous. To protect against the effects of random mutations, eukaryotic cells have DNA proofreading enzymes that reduce the error rate 1000 fold to 1 in 100 million. Since DNA viruses follow molecular dogma exactly and use the host s replication machinery just as the host would, they can also take advantage of the host s proofreading enzyme. As a result DNA viruses have a relatively low rate of mutation. In contrast, the host doesn t have a proofreading enzyme that corrects for errors when RNA is made into RNA, because it never is in eukaryotic cells. As a consequence RNA viruses cannot use a host proofreading enzyme when they copy their RNA into RNA. They also don t bring along their own proofreading enzymes, so mutations in RNA viruses are quite high. But RNA viruses have developed strategies to counter the potential disadvantages of a high rate of mutation: Mutation rates always occur as a number of mutations per length of DNA (normally 1 in 100,000 as we saw above). The longer the stretch of RNA to be replicated, the more mutations will occur. The two strategies RNA viruses have come up with are: First, to reduce the size of the genome as much as possible. The second is to divide their genomes into small pieces (called segmentation). Each piece of a segmented RNA virus genome is a lot smaller than DNA viruses that aren t segmented, therefore the error rate will be reduced. Segmented genomes confer evolutionary advantages that are also related to pathogenesis. If different strains of a virus with a segmented genome infect the same cell they can can shuffle and combine genes and produce offspring with unique characteristics. This process is called antigenic shift. Fig 4.4.1: A diagram of the influenza virus. It is an RNA virus and its genome is segmented into 8 pieces. This How would the high error rate of replication seen in RNA viruses contribute to pathogenicity? Lesson 4.4 reduces the error rate for mutation. 6
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