H2 Biology - Seminar Notes (Genetics of Viruses) PDF

H2 Biology – Seminar Notes [Genetics of Viruses] Name: Class: Date: CORE IDEA 2: GENETICS AND INHERITANCE G...

H2 Biology – Seminar Notes [Genetics of Viruses] Name: Class: Date: CORE IDEA 2: GENETICS AND INHERITANCE GENETICS OF VIRUSES Your syllabus requires you to: Core Idea 1 (e) describe the structural components of viruses, including viruses and bacteriophages, and interpret drawings and photographs of them (f) discuss how viruses challenge the cell theory and concepts of what is considered living Core Idea 2 (d) Describe the structure and organisation of viral, prokaryotic and eukaryotic genomes (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns) (e) Describe how the genomes of viruses are inherited through outlining the reproductive cycles of: i) bacteriophages that reproduce via a lytic cycle, e.g. T4 phage; ii) bacteriophages that reproduce via lytic and lysogenic cycle, e.g. lambda (λ) phage; iii) an enveloped virus, e.g. influenza; and iv) retroviruses, e.g. HIV. (f) Describe how variation in viral genomes arises, including antigenic shift and antigenic drift. 2024 1  H2 Biology – Seminar Notes [Genetics of Viruses] 1. Viruses: Obligate intracellular parasites LO: (e) describe the structural components of viruses, including viruses and bacteriophages, and interpret drawings and photographs of them (f) discuss how viruses challenge the cell theory and concepts of what is considered living In the late 1800s, scientists studying stunted tobacco plants discovered a new kind of disease-causing agent, or pathogen. It was so small that it passed through screens that filtered out bacteria, and it could not be seen with a light microscope. The scientists called this unseen infectious entity a virus, a term that means “poison” in Latin. Structure of viruses Viruses range in size from 20 to 400 nm in diameter. o In comparison, a typical bacterial cell is 1,000 nm in diameter, and the diameter of most eukaryotic cells is 10 to 1,000 times that of a bacterial cell. Viruses differ greatly in their host range – the number of species and cell types they can infect. Viruses are parasites and are dependent on the host cells for most of their requirement, including o building blocks such as amino acids and nucleosides; o protein-synthesising machinery o energy, in the form of adenosine triphosphate. Figure 1 – Diversity of viral structures (Reece J. B., et al., 2014) i. Genome Unlike eukaryotic and bacterial cells which contain double-stranded DNA genomes, viral genomes can be: single-stranded DNA, double-stranded DNA, single-stranded RNA or double-stranded RNA. The genome is usually organized as a single linear or circular molecule of nucleic acid. 2024 2  H2 Biology – Seminar Notes [Genetics of Viruses] ii. Capsid All viruses have a protein coat called a capsid, which encloses the genome. Capsids are composed of one or several different protein subunits called capsomeres. Capsids have a variety of shapes: helical, polyhedral. The capsid functions to o protect nucleic acids from digestion by nucleases, and o facilitate entry of viral genome into host cell by containing sites for attachment to host cell. iii. Envelope Many viruses that infect animals have a viral envelope (lipid bilayer) enclosing the capsid. The viral envelope is acquired as the virus leave the host cell via budding. o Hence, the viral envelope is made up of host cell phospholipids host membrane proteins derived from the host cell surface membrane, and unique viral glycoproteins encoded by viral genome. The viral envelope functions to: o facilitate entry of viruses into host cells as the viral glycoproteins recognise and bind to specific receptors on the host cell surface membrane, and o avoid recognition by the host defence mechanisms. a. Challenges to cell theory and concepts of living Two of the three tenets of the cell theory pertain to life and living. But it is difficult to define ‘life’. See Figure 2 Lam & Lam, 2014 Reece, et al., 2014 Mason, et al., 2017 Freeman, et al., 2017 Cells Order Cellular organisation Cells Nutrition Energy processing Ordered complexity Replication Respiration Evolutionary adaptation Sensitivity Evolution Excretion Regulation Growth, development & (genetic) Information Growth & development Growth & development reproduction Energy Movement Response to the Energy utilization Reproduction environment Homeostasis Sensitivity Reproduction Evolutionary adaptation Adaptability Figure 2 -Characteristics of life identified in various textbooks of biology 2024 3  H2 Biology – Seminar Notes [Genetics of Viruses] Let’s consider how viruses match up against some of characteristics of life Characteristic of life “living” “non-living” Cellular organisation Viruses are not made up of cells. Ordered complexity Viruses have capsids that are highly ordered structures. Energy processing/ utilisation Viruses direct its host cells to Viruses cannot generate or provide the energy needed to store energy. Outside of the produce more viral particles. host cell, viruses have no need for energy. Growth & development Viruses do not grow in size nor complexity. Reproduction Viruses carry their own genetic Viruses cannot reproduce material. outside of the host cell. Viruses are able to reproduce Viruses are not formed directly by taking over the host cell’s from a pre-existing virus. They machinery to make more viral are assembled from ‘mass- particles. produced’ components in the host cell. Evolutionary adaptation Viruses have high mutation rates, which create genetic variation, allowing some viruses to survive in unfavourable environments Sensitivity/ Response to Viruses may respond to Viruses do not respond to environment environmental stimuli and stimuli outside a host cell. transit between different Many viruses do not have a phases of the reproductive latency phase in their cycle e.g. lysogenic to lytic reproductive cycles. cycle in λ phage, latency and reactivation in HIV. Regulation/ Homeostasis Viruses do not maintain internal conditions that are different from their environment. 2024 4  H2 Biology – Seminar Notes [Genetics of Viruses] 2. Nature of Viruses Figure 3 - Schematic representation of viral particle (Madigan, Bender, Buckley, Sattley, & Stahl, 2019) a. Viruses consist of a nucleic acid genome packaged in a protein coat. The simplest definition of a virus is an infectious nucleic acid surrounded by protein. The complete, infectious extracellular virus particle is called a virion. The virion of any virus includes a protein shell, called a capsid, and the viral genome. Most bacterial viruses are naked, with no further layers. Many animal viruses have an envelope (See Figure 3) that consists of a phospholipid bilayer (taken from the host cell membrane) and viral proteins. In enveloped viruses, the inner structure of nucleic acid plus capsid is called the nucleocapsid. b. Virus genomes are either RNA or DNA, but not both. The viral genome encodes proteins that enable it to replicate and to be transmitted from one cell to another, and from one organism to another. The viral genome consists of either RNA or DNA. o A few unusual viruses use both RNA and DNA as genetic material but at different stages of the reproductive cycle of the virus, such as retroviruses. RNA genomes are typically smaller than DNA genomes. See Figure 4. The viral genome may be linear or circular. The viral genome may be single-stranded or double-stranded. Single-stranded RNA (ssRNA) genomes are further characterised into positive-sense single- stranded RNA or negative-sense single-stranded RNA genomes. See Figure 5. o Positive-sense single-stranded RNA: genome has identical sequence to the mRNA used to make viral proteins. So, the genome can be used directly for translation. o Negative-sense single-stranded RNA: genome has complementary sequence to the mRNA used to make viral proteins. So, the genome cannot be used directly for translation. 2024 5  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 4 - Sizes of viral genomes (Madigan, Bender, Buckley, Sattley, & Stahl, 2019) [Note E.coli, HIV, Lambda and T4 for comparison] Figure 5 - Implications of ssRNA on viral reproduction (Wei, 2013) (A) Positive-sense ssRNA can be used directly as template for synthesis of viral proteins. (B) Negative-sense ssRNA needs to be transcribed into mRNA, which is used as template for synthesis of viral proteins 2024 6  H2 Biology – Seminar Notes [Genetics of Viruses] c. Viruses can replicate only within living cells. They are obligate/ obligatory intracellular parasites. They rely on the host cell for basic building blocks, energy, and protein synthesis. o They lack enzyme systems that produce the basic chemical building blocks of life: nucleotides, amino acids, carbohydrates, and lipids. o They lack enzyme systems that generate usable chemical energy (in the form of ATP) by photosynthesis or metabolism of sugars and other molecules. o They lack ribosomes, transfer RNAs, and the associated enzymatic machinery that directs protein synthesis. They may carry their own unique enzymes that are necessary for their reproductive cycles. o Some bacteriophages contain an enzyme that resembles lysozyme, which is used to ‘make a hole’ in the peptidoglycan layer to allow nucleic acid to enter the cytoplasm of the bacterial cell. A similar protein is produced in the later stages of infection to lyse the host cell. o Retroviruses, such as HIV contain reverse transcriptase (RNA-dependent DNA polymerase) needed to make new virions via DNA intermediates. 2024 7  H2 Biology – Seminar Notes [Genetics of Viruses] 2.1 General reproductive cycle of viruses a. Attachment (Adsorption) Viral surface proteins recognise and bind to specific receptors (proteins, carbohydrates, or lipids) on the host cell surface. The host cell receptor may be found on cells of certain species or certain cell types. This determines the host range of the virus. b. Entry and uncoating (Acheson, 2011) Many bacteriophages have specialised tails that drill holes in cell wall and membranes to enable passage of DNA genome into host cell cytoplasm. Plant viruses often penetrate (means enter) because of cell wall damage caused by abrasion or wound caused by insects. Some animal viruses penetrate host cells by fusing their lipid envelope with host cell surface membrane. See Figure 6. Many animal viruses enter host cells through receptor mediated endocytosis. See Figure 6. o Receptor mediated endocytosis is different from phagocytosis. o Phagocytosis refers to ingestion of relatively large particles (>0.5m in diameter). o In animals, phagocytosis is carried out primarily by specialised cells such as macrophages, neutrophis and dendritic cells. (to be covered in Extension topic A) Upon penetration, uncoating occurs whereby the capsid proteins are degraded, and the viral nucleic acids are released into the cytoplasm. c. Synthesis Viruses make use of host cellular machinery, such as ribosomes and enzymes required in the post-translational processing of proteins, to synthesise viral proteins and replicate viral genome. d. Assembly Assembly involves bringing together newly formed viral genome and viral proteins to form the nucleocapsid. e. Release Non-enveloped, naked viruses (eg. bacteriophages) causes the host cell to lyse and release the newly synthesised viral particles. Enveloped viruses are usually released via budding. Viral proteins and/or glycoproteins are inserted onto the host cell surface membrane. Nucleocapsids bind to the regions of the host cell surface membranes studded with these proteins, and bud off the host cell. 2024 8  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 6 – Animal virus entry (Willey, Sherwood, & Woolverton, 2017) 2024 9  H2 Biology – Seminar Notes [Genetics of Viruses] 3. Genetics of T4 phage LO: (d) Describe the structure and organisation of viral, prokaryotic and eukaryotic genomes (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns. (e) Describe how the genomes of viruses are inherited through outlining the reproductive cycles of: (iii) bacteriophages that reproduce via lytic cycle only, including T4 phage a. Structure of virion Figure 7 – Structure of T4 phage (Snustad & Simmons, 2012) The head of T4 phage is a prolate icosahedral capsid assembled from capsomeres. It contains the viral genome inside. Figure 8 – Relation between capsid vs capsomere (Tao, et al., 2018) The hollow tail core provides the channel through the phage DNA is injected into the bacterium. The tail sheath pushes the tail core through the bacterial cell wall when it contracts. The six tail fibers are used to locate receptors on host bacterial cell. The tail pin binds to receptors. 2024 10  H2 Biology – Seminar Notes [Genetics of Viruses] b. Structure and organisation of T4 phage genome The T4 phage has double-stranded DNA (dsDNA) genome that is 169kb in size. [1kb equals 1000 nucleotide (base) pairs] The DNA inside T4 phage virion is linear, but it can circularise during infection. The T4 phage genome contains 288 protein-coding genes. (Brooker, 2018) c. Reproductive cycle of T4 phage T4 phage is a lytic phage/ virulent phage. o This means when it infects a bacterium, it replicates and kills the host cell. o The genome of a virulent phage is not capable of integration into a host chromosome. T4 phage is an obligate lytic phage of Escherichia coli bacteria. i. Attachment o T4 phage long tail fibres recognise and bind to specific receptor sites on the outer surface of the host bacterial cell. ii. Entry o T4 phage tail sheath contracts. o T4 phage tail lysozyme complex forms pore in the cell wall by digesting the peptidoglycan layer. o T4 phage dsDNA is injected into the host bacterial cell, leaving an empty capsid outside. Figure 9 – Attachment and entry stages of T4 phage reproductive cycle (Madigan, Bender, Buckley, Sattley, & Stahl, 2019) 2024 11  H2 Biology – Seminar Notes [Genetics of Viruses] iii. Synthesis o Host cell machinery is used for synthesis of phage proteins and replication of phage DNA. For example bacterial RNA polymerase is used for transcription of phage DNA to make mRNA, while bacterial ribosomes are used for translation to make phage proteins. o (Early) gene expression directs the synthesis of phage proteins that shut off the transcription, translation, and replication of bacterial genes, and synthesis of early proteins. o Some proteins, such as T4 nucleases hydrolyse host bacterial DNA. ▪ [How does viral DNA avoid being degraded?] In T4 phage, the cytosines in the DNA are replaced by hydroxymethycytosine, a modified base that does not occur in E.coli. This protects the T4 DNA from degradation by T4 nucleases used to degrade host DNA. o (Middle) gene expression directs the synthesis of structural components needed for assembly of phage particles. o Some proteins, such T4 DNA polymerase catalyse replication of phage DNA. o (Late) gene expression directs the synthesis of proteins needed for release of assembled phage particles, such as phage lysozyme. Figure 10 – Synthesis and assembly stages of T4 phage reproductive cycle (Madigan, Bender, Buckley, Sattley, & Stahl, 2019) [absolute time is not important] iv. Assembly o Three separate sets of proteins self-assemble to form phage heads, tails, and tail fibres. o The phage genome is packaged inside the capsid as the head forms. v. Release o Phage lysozyme damages the bacterial peptidoglycan cell wall, allowing surrounding fluid to enter the cell. o The host cell swells and lyses via osmotic lysis, releasing new phage particles. 2024 12  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 11 - Reproductive cycle of T4 phage (Reece, et al., 2014) 2024 13  H2 Biology – Seminar Notes [Genetics of Viruses] 4. Genetics of lambda () phage LO: (d) Describe the structure and organisation of viral, prokaryotic and eukaryotic genomes (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns. (e) Describe how the genomes of viruses are inherited through outlining the reproductive cycles of: (ii) bacteriophages that reproduce via lytic and lysogenic cycle, including lambda () phage; a. Structure of  phage virion Figure 12 - Structure of  virion with tail tip fibre Figure 13 - Electron micrograph of  virion with (Snustad & Simmons, 2012) tail tip fibre and side tail fibres (Knipe & Howley, 2013) The head of  phage is an icosahedral capsid. The  phage has a non-contractile tail. The tail fibres of  phage may vary: o All  phage virions have the central tail tip fibre. o Some  phage virions have side tail fibres, some do not. b. Structure and organisation of  phage genome The λ phage has double-stranded DNA (dsDNA) genome that is 48.5kb in size. The DNA inside λ phage virion is linear, but it can circularise during infection. The λ phage genome contains 36 protein-coding genes. (Brooker, 2018) 2024 14  H2 Biology – Seminar Notes [Genetics of Viruses] c. Reproductive cycle of  phage λ phage is a temperate phage that can follow a lytic cycle or lysogenic cycle. o Environmental conditions influence the integration of viral DNA into a host chromosome and how long the virus remains in the lysogenic cycle. o If nutrients are readily available, the phage usually proceeds directly to the lytic cycle. λ phage infects Escherichia coli bacteria. i. Attachment o The λ phage uses its tail fibre to bind to specific receptor sites on the outer surface of a bacterial cell. ii. Entry o The λ phage make use of specific pores in the cell surface of bacterium to inject its DNA into the host bacterial cell, leaving an empty capsid outside. o Inside the host bacterial cell, the λ DNA circularises – the two ends of linear DNA become covalently attached to each other. o The λ phage enters either the lytic or lysogenic cycle, depending on the metabolic state of the host bacterial cell. ▪ If the host bacterial cell is metabolically active, the λ phage would most likely enter the lytic cycle. And vice versa. Integrase catalyses the integration of the lambda DNA into the host DNA in the form of a prophage Figure 14 - Reproductive cycle of lambda phage (Reece, et al., 2014) 2024 15  H2 Biology – Seminar Notes [Genetics of Viruses] Enters lysogenic cycle iii. Integration o The gene coding for integrase in the λ phage genome is expressed soon after entry. o Integrase recognises specific DNA sequences known as attachment sites and catalyses the integration of the λ DNA into the host DNA via site-specific recombination. See Figure 15 for illustration of how integrase works. o The integrated phage DNA in a bacterial chromosome is called a prophage. o A prophage gene codes for a repressor protein that suppresses transcription of most of the other prophage genes, such as proteins involved in virion assembly and lysis of host cells. o As a prophage, the  may remain latent/ dormant for many generations. iv. Prophage multiplication o The prophage DNA is replicated alongside bacterial DNA as every time the infected E. coli cell prepares to divide via binary fission. As a result, prophage DNA is passed on to the daughter bacterial cells (which will also be infected). o A single infected bacterium can thus quickly give rise to a large population of bacteria carrying the virus in the prophage form. This mechanism enables the phage to propagate without killing the host cells on which they depend. Switches to lytic cycle: About once in every 105 cell divisions, the prophage spontaneously excises from host chromosome. The prophage may be also induced to transit from the lysogenic cycle to the lytic cycle by environmental signals, such as radiation, availability of nutrients, or the presence of certain chemicals. v. Induction o The prophage is excised from the host chromosome, forming a circular DNA. vi. Synthesis o The ‘early’ genes direct synthesis of proteins that mediate phage-specific DNA replication. o These proteins direct host machinery to replicate phage DNA. o The phage DNA is replicated via rolling-circle mechanism. o The ‘late’ genes direct synthesis of proteins involved in lysis, assembly of head and tail subunits of virion. vii. Assembly o Three separate sets of proteins self-assemble to form phage heads, tails, and tail fibres. o The circular phage DNA is cut at the cohesive sites to yield the linear dsDNA (with cohesive ends) which is then packaged into viral particles. o The phage genome is packaged inside the capsid as the head forms. viii. Release o Phage lysozyme damages the bacterial peptidoglycan cell wall, allowing surrounding fluid to enter the cell. o The host cell swells and lyses via osmotic lysis, releasing new phage particles. 2024 16  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 15 – Integration of λ DNA into the E. coli chromosome (Brooker, 2018) 2024 17  H2 Biology – Seminar Notes [Genetics of Viruses] Concept Check! 1. Compare the reproductive cycles of T4 and  phages. 2024 18  H2 Biology – Seminar Notes [Genetics of Viruses] 5. Genetics of enveloped virus (influenza virus) LO: (d) Describe the structure and organisation of viral, prokaryotic and eukaryotic genomes (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns. (e) Describe how the genomes of viruses are inherited through outlining the reproductive cycles of: (iii) enveloped viruses, including influenza virus; a. Structure of influenza virus Influenza viruses are enveloped viruses. There are three types of influenza viruses: influenza A (most virulent), influenza B and influenza C. Our syllabus focuses on influenza A. Figure 16 - Structure of influenza A virus (Krammer, et al., 2018) Influenza A virus is zoonotic pathogen that can infect a broad range of species. o A zoonosis is an infectious disease that has jumped from a non-human animal to humans. Haemagglutinin (HA) and neuraminidase (NA) are two major viral envelope glycoproteins that recognise sialic acid on host cells. Influenza A viruses are classified into o 18 HA subtypes (H1 to H18) o 11 NA subtypes (N1 to N11) HA proteins exhibit specific binding affinities for the different sialic acid-linked glycoproteins expressed on cell surface. o [How are the viruses specific for the host?] For example, avian viruses preferentially bind to -2,3-linked sialic acid receptors, while human viruses preferentially bind to -2,6-linked sialic acid receptors. 2024 19  H2 Biology – Seminar Notes [Genetics of Viruses] b. Structure of influenza viral genome The influenza virus has negative-sense, single-stranded RNA (ssRNA) genome that is 13.5kb in size. The influenza viral genome is linear. The influenza viral genome is segmented, which means the complete genome is divided two or more physically separate molecules of nucleic acid. Influenza A virus genome consists of 8 segments. o Each segment contains one to a few genes. o Each segment is folded upon itself and associates with nucleocapsid proteins/ nucleoproteins to form helical nucleocapsid. See Figure 16. The influenza viral genome contains 11 protein-encoding genes. (Brooker, 2018) c. Reproductive cycle of influenza A virus i. Attachment Haemagglutinin recognises and binds to sialic acid receptors on host cell. Figure 17 - Reproductive cycle of influenza A virus (Krammer, et al., 2018) The following stages of reproductive cycle are described with reference to this image. ii. Entry Virus enters the host cell via receptor-mediated endocytosis. o The host cell surface membrane invaginates, surrounds the virus, and pinches off, forming an endosome. Endosomal release is triggered by acidification of endosome. o This triggers fusion of viral envelope with endosome membrane, releasing RNP complexes and influenza viral enzymes into the cytoplasm. 2024 20  H2 Biology – Seminar Notes [Genetics of Viruses] iii. Synthesis RNP (ribonucleoprotein) complexes are imported into the nucleus through nuclear pores. Viral RNA-dependent RNA polymerase uses the negative-sense ssRNA as a template to make: o complementary positive-sense RNA (represented as cRNP) which is used as a template to make more copies of negative-sense RNA (represented as vRNP). [This is akin to replication of viral genome.] o complementary positive-sense RNA which is used as mRNA (represented as mRNA). [This is akin to transcription.] Viral mRNAs are exported to the cytoplasm through nuclear pores. o Capsid proteins (represented as NP) and viral enzymes (represented as coloured shapes) are synthesised by ribosomes in the cytosol. o Glycoproteins (HA and NA) are synthesised by ribosomes bound to the rER, inserted into the rER membrane, and then modified in the Golgi apparatus (glycosylation). Viral proteins that are needed for replication and assembly are imported into the nucleus. iv. Assembly New RNP complexes are assembled in the nucleus and exported to the cytoplasm. v. Release New influenza virus buds from the infected host cell, with the RNP complexes enveloped by the host cell surface membrane studded with viral HA and NA glycoproteins. Neuraminidase cleaves the sialic acid residues on host cell, facilitating the release of the newly formed virus from the host cell. This prevents the aggregation of new viruses to the host cell. Figure 18 – Release stage of influenza virus reproductive cycle (Willey, Sherwood, & Woolverton, 2017) 2024 21  H2 Biology – Seminar Notes [Genetics of Viruses] 6. Genetics of retrovirus (HIV) LO: (d) Describe the structure and organisation of viral, prokaryotic and eukaryotic genomes (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns. (e) Describe how the genomes of viruses are inherited through outlining the reproductive cycles of: (iv) retroviruses, including HIV a. Structure of HIV Figure 19 - Structure of HIV (Brooker, 2018) HIV is an enveloped virus with a conical capsid. HIV carries unique viral proteins reverse transcriptase, integrase and protease that are needed following entry into host cell. Reverse transcriptases do not have efficient proofreading mechanisms to remove incorrectly incorporated nucleotides in proviral DNA. This accounts for high rates of mutations. Reverse transcriptase has two catalytic functions: RNA-dependent DNA polymerase and DNA- dependent DNA polymerase The envelope gene, which encodes the proteins that bind to CD4 and the co‑receptors, is able to withstand extensive mutations. 2024 22  H2 Biology – Seminar Notes [Genetics of Viruses] b. Structure of HIV genome The human immunodeficiency virus has positive sense, single-stranded RNA (ssRNA) genome that is 9.7kb in size. o This means that the genome can be used directly for synthesis of viral proteins. The HIV genome is linear. The HIV genome consists of two identical RNA molecules. Each RNA molecule associates with nucleocapsid proteins to form nucleocapsids. c. Reproductive cycle of HIV i. Attachment Glycoprotein gp120 recognise and bind to CD4 receptors on CD4+ T-cells. o This induces conformational change in gp120, exposing regions that interact with CCR5 or CXCR4 chemokine receptor. See Figure 20. Figure 20 – Attachment and entry stages of HIV reproductive cycle (Tortora, Funke, & Case, 2019) [for stepwise illustration of entry stage, not for memorisation] ii. Entry Attachment causes change in glycoprotein gp41 which leads to the fusion of the viral envelope with the host cell surface membrane. The conical capsid is digested by cellular enzymes, releasing the viral (+) ssRNA and viral enzymes into the cytoplasm. This is known as uncoating. (Figure 21, step 6) 2024 23  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 21 – Reproductive cycle of HIV (Engelman & Cherepanov, 2012) iii. Reverse transcription & Integration (Figure 21, steps 4 to 6) RNA-dependent DNA polymerase activity of reverse transcriptase catalyses the synthesis of a single (-) ssDNA strand complementary to the viral (+) ssRNA genome. The viral RNA is then degraded. DNA-dependent DNA polymerase activity of reverse transcriptase then catalyses the synthesis of a complementary (+) ssDNA strand complementary to the first, forming a viral dsDNA molecule. Integrase catalyses the insertion of proviral DNA into host chromosome. o The provirus remains permanently inside the host genome. iv. Synthesis (Figure 21, steps 7 & 9) Under the presence of certain environmental triggers, the proviral genes are transcribed into (+) ssRNA molecules by the host cell’s DNA-dependent RNA polymerase. The (+) ssRNA molecules are exported out of the nucleus into the cytoplasm. Full length (+) ssRNA molecules serve as HIV viral genomes for the next viral generation. Some (+) ssRNA molecules also serve as mRNA templates for translation into new viral proteins. o Viral polyprotein consisting of capsomeres (capsid proteins) and viral enzymes (reverse transcriptase, integrase, protease) are synthesised by free ribosomes in the cytosol. Note: viral proteins and enzymes are synthesised as a continuous polypeptide called polyprotein, which is later cleaved into smaller functional products by viral protease during maturation. o Glycoproteins (gp120 and gp41) for the viral envelope are synthesised by ribosomes bound to the RER, inserted into the RER membrane, and then modified in the Golgi apparatus. Vesicles transport glycoproteins to the cell surface membrane. 2024 24  H2 Biology – Seminar Notes [Genetics of Viruses] v. Assembly (Figure 21, step 10) The newly-synthesised viral (+) ssRNA genome are packaged with viral polyproteins near the cell surface membrane. vi. Release & Maturation (Figure 21, steps 11 to 13) Each new HIV virus buds from the infected host cell, surrounded by the host cell surface membrane studded with viral glycoproteins gp120 and gp41. Viral protease cleaves the HIV polyproteins into smaller functional products (e.g. reverse transcriptase, integrase, capsomere) to form the mature HIV virus. Figure 22 – Release stage of HIV reproductive stage (Brooker, 2018) [for stepwise illustration of release & post-release maturation, not for memorisation] 2024 25  H2 Biology – Seminar Notes [Genetics of Viruses] Concept Check! 1. Compare the reproductive cycles of  phage and HIV. 2. Compare the structure and organisation of genomes for T4 phage,  phage, influenza virus and HIV. 2024 26  H2 Biology – Seminar Notes [Genetics of Viruses] 7. Variation in viral genomes LO: (f) Describe how variation in viral genomes arises, including antigenic shift and antigenic drift. a. Mechanisms resulting in genetic variation i. Mutations Figure 23 - Comparison of viral mutation rates (Duffy, Shackelton, & Holmes, 2008) Mutation rate refers to the number of genetic errors (point mutations, insertions and deletions) that accumulate per unit time, or per generation (for obligately lytic viruses, per burst), or per round of genomic replication. Mutations may be caused by polymerase errors, nucleotide base modifications caused by other cellular enzymes. RNA viruses (which utilize RNA-dependent RNA polymerases) mutate faster than retroviruses (with RNA-dependent DNA polymerases or reverse transcriptases), which mutate faster than DNA viruses (with DNA polymerases). RNA-dependent RNA polymerases and RNA-dependent DNA polymerases are more error- prone than DNA polymerases. It is well known that influenza viral RNA-polymerase lacks proofreading function. In contrast, DNA polymerases can contain proofreading domains, which further reduce the mutation rate during DNA replication. ii. Genetic reassortment This refers to the exchange of genetic material in the form of RNA segments between genotypically different viruses with segmented genomes. This results in the formation of a hybrid genome. This assumes two or more virus strains co-existing in host cell. Two of the three major human influenza pandemics in the twentieth century (1957 and 1968) and this century (2009) were due to the re-assortment between the human influenza A virus and other host species influenza virus. iii. Genetic recombination This refers to the exchange of genetic material between different viral strains. This results in the formation of a hybrid genome. This assumes two or more virus strains co-existing in host cell. Genetic recombination has been observed in HIV, where patients infected with more than one genetically distinct HIV resulted in hybrid viruses in their viral populations. For example, a recombinant derived from HIV-1 subtypes A and E has resulted in an epidemic in Thailand in 1989. 2024 27  H2 Biology – Seminar Notes [Genetics of Viruses] b. Evolution of influenza viruses i. Antigenic drift This phenomenon is caused gradual accumulation of nucleotide mutations and amino acid substitutions in the HA and NA surface glycoproteins within each virus subtype, resulting in emergence of new antigenic variants. This usually involves subtle genetic changes. This phenomenon necessitates frequent updates of influenza vaccines to ensure sufficient antigenic relatedness between the vaccine and emerging virus variants. See Figure 25a. ii. Antigenic shift This phenomenon is caused by genetic reassortment or genetic recombination, resulting in emergence of new antigenic pattern. This usually involves drastic genetic changes. This phenomenon is associated with influenza A pandemics. Figure 24 - Impact of antigenic shift and antigenic drift on influenza virus structure (Krammer, et al., 2018) 2024 28  H2 Biology – Seminar Notes [Genetics of Viruses] Figure 25 – Evolution of influenza viruses and impact on human health (Madigan, Bender, Buckley, Sattley, & Stahl, 2019) (a) Antigenic drift reduces efficacy of vaccines as new surface antigens appear from mutations in genes. (b) Coinfection of zoonotic host can lead to emergence of new virus that infect humans. viruses may damage or kill cells by causing the release of hydrolytic enzymes from lysosomes. some viruses may cause infected cells to produce toxins that lead to disease symptoms others have envelope proteins that are toxic. vaccines are harmless derivatives of pathogenic microbes that stimulate the immune system to mount defenses against the actual pathogen vaccines can prevent certain illnesses 2024 29