Viruses - H2 BIOLOGY 2016-2017

Viruses may be​ living ​organisms because: a) they can 'reproduce' (replicate) in their intracellular state Viruses are ​nonliving​ organisms because they: a)​ ​lack cellular organelles​ though they have a protein capsid enclosing a nucleic acid core & sometimes a viral envelope (Cell theory: all living organisms are composed of cells) b) ​are ​unable to carry out​ metabolism, do not require nutrition and neither grow or excrete etc Viruses are ​obligate parasites​ because: a) ​requires host cell​ to complete its reproductive cycle b) they ​lack cellular structures​ such as : - enzymes for most metabolic processes - protein synthesizing machinery ( ribosomes, RNA polymerases) - energy in the form of ATP to drive replication, transcription, translation and cellular metabolism - building blocks such as DNA/RNA and amino acids and hence, have to​ hijack host cell metabolic machinery f​ or its replication, transcription and translation. Basic structure of a virus: a) Viral genome- ​1. contains either RNA o ​ r​ DNA 2. can be circular or linear 3. can be double-stranded (ds) or single-stranded (ss) b) Capsid- ​1. a protein coat which encloses the viral genome 2. comprises capsomeres 3. various shapes (icosahedral, helical, spherical) c) Envelope- ​1) derived from the host CSM when the virus b ​ uds​ off 2) contains viral glycoproteins which help the virus attach itself to the host cell 3) protects the viral genome from the effects of various genomes & chemicals d) Other proteins such as reverse transcriptase (for animal viruses) e) tail pins and tail fibres (for bacteriophages) Comparing and contrasting between structures of bacteriophages and animal viruses: 1. Presence/absence of a ​viral envelope 2. presence/absence of ​glycoproteins 3. presence/absence of ​tail pins/tail fibres 4. ssRNA​ genome vs ​dsDNA​ genome

Viral Infection: Temperate phages​: bacteriophages which are capable of a lysogenic life cycle Lytic phages​: bacteriophages which undergoes the lytic life cycle Lytic​- reproductive cycle that involves the l​ ysis of the host cell Lysogenic​- reproductive cycle that involves the i​ ntegration of the viral genome into the host genome​. The viral genome is replicated as host genome is replicated.

Comparison

Lytic (T4)

Lysogenic (Lambda)

Purpose

For m ​ ass reproduction of viruses

For ​integration into host genome​ and to remain dormant ​until appropriate environmental stimulus triggers mass reproduction

Gene Expression

Genes coding for structural components​ such as capsid and enzymes such as lysozymes are synthesized for the ​assembly of new viruses

Genes coding for a repressor protein and an integrase​ are expressed to allow for the phage genome to be integrated into host genome​.

Reproduction of virus

Newly assembled phages formed are ready​ and will be released from the host cell

No new viruses are formed. Instead, viral DNA replicates as part of the host genome.

Replication of viral genome

Replication of phage DNA occurs u ​ sing the host cell DNA polymerase​.

Viral DNA replicates as part of the host genome ​each time the host cell divides​.

Fate of host

Host cell d ​ ies upon the release​ of new phages

Host cell ​survives​ and all the daughter cells will contain the viral genome as part of their genomes

Viral reproductive cycle consists of mainly: ​adsorption, penetration, replication, maturation and​ release. T4 reproductive cycle: Adsorption:​ ​Tail fibres of T4 phage recognises and binds to specific receptor sites on the host bacterium surface. The base plate comes into contact with the surface of the host bacterium cell.

Penetration​: The base plate and tail sheath then undergoes conformational change. The tail sheath contracts to drive the tail tube through a hole in the bacterial cell wall (degraded by lysozyme). The phage DNA is injected into the cytoplasm while the empty capsid is left outside the cell. Replication: ​The phage DNA directs the synthesis of phage proteins such as lysozymes, tail and capsid proteins using the host cell RNA polymerases (for transcription) and ribosomes (for translation). Lysozymes produced is used to degrade the host cell DNA and halt the host macromolecular synthesis and replication processes. Viral DNA is replicated using the host cell replication machinery such as nucleic acids. Maturation: ​The capsid proteins spontaneously self-assemble around the phage DNA to form new phages. Release:​ The phage-encoded lysozyme is released in the host cell, causing osmotic lysis of the host cell and the release of new phages.

Lambda reproductive cycle: Adsorption:​ Tail fibres of T4 phage recognises and binds to specific receptor sites on the host bacterium surface. The base plate comes into contact with the surface of the host bacterial cell. Penetration:​ The base plate and tail sheath undergoes a conformational change. The phage DNA is injected into the cytoplasm while the empty capsid is left outside the cell. Integration:​ The phage DNA circularises and is integrated into a specific site of the bacterial chromosome via integrase to become a non-infectious prophage. Prophage replication:​ 1 prophage gene codes for repressor protein that prevents transcription of most of the other prophage genes. Thus, the phage genome is mostly silent within the bacterium. Each time the host cell divides, phage DNA replicates as part of the host bacterial DNA, and every daughter bacterium now contains a prophage. Activation:​ An environmental signal can trigger the destruction of repressor proteins. Repressor proteins are no longer made and phage genome is excised from bacterial DNA in a few bacteria. Replication:​ Phage genes are activated. Phage components are made using the host bacterium’s machinery like DNA polymerase for replication of phage DNA, and RNA polymerases and ribosomes for synthesis of new phage proteins.

Maturation: ​New phages are self-assembled spontaneously, with capsid proteins surrounding phage genomes. Release: ​A phage-encoded enzyme breaks down the bacterial cell wall, causing osmotic lysis and release of intact lambda bacteriophages.

Note: Glycoproteins are translated in​ RER​ while capsid proteins and other viral enzymes (HIV protease) are translated by ​free ribosomes​. Influenza reproductive cycle: Adsorption:​ Haemagglutinin (HA) on viral envelope binds to sialic acid receptors on the host cell membrane.

Penetration: ​After binding, host cell surface membrane invaginates and places the virus in an endosome. The lowered pH in the endosome triggers a conformational change of the HA, causing fusion of endosomal membrane and viral envelope, releasing the nucleocapsid into the cytoplasm. The cellular enzymes remove the capsid, virus then dispatches its genetic material and internal proteins into nucleus. Replication:​ Viral genome acts as a template for synthesizing complementary RNA strands via RNA-dependent RNA polymerases. Complementary RNA strands then served as templates for making new RNA genome OR mRNA for producing viral proteins such as capsid proteins and glycoproteins using host cell’s ribosomes. Maturation: ​Glycoprotein HA and enzyme NA transported via vesicles to host cell surface membrane. Capsid proteins transported back into the nucleus for self-assembly with viral genome. Mature viruses then transported to the host cell surface membrane where HA and NA had been incorporated into. Release:​ Mature viruses bud off from the host cell covered with an envelope derived from the host cell surface membrane which has viral HA and NA inserted. Sialic acid receptors are cleaved off the surface of the new viral particles through viral NA, and this facilitates the release of viral particles from the host cell.

HIV reproductive cycle: Adsorption: ​GP120 on the viral envelope binds to CD4 receptors and co-receptors CCR5 on the host cell membrane. Penetration: ​Binding of GP120 to the host receptors triggers conformational change in GP41, causing viral envelope to fuse with host cell surface membrane, and the virus releases its nucleocapsid into the cytosol. The capsid is degraded, releasing enzymes such as integrase and reverse transcriptase as well as RNA into cytoplasm. Reverse Transcription: ​Reverse transcription of RNA genome into a single-stranded DNA via Reverse transcriptase. A second complementary DNA is then synthesized by Reverse transcriptase to form a linear double-stranded DNA copy of the original genome. Integration: ​Viral DNA is integrated into the host DNA through reactions catalysed by integrase, forming provirus (remains latent for years and undergoes retroviral replication) Provirus activation:​ due to extracellular stimuli, there’s an activation of provirus which forces the cell to produce more virions. Replication: ​Proviral genes (DNA) is transcribed to produce copies of RNA. Transcribed RNA serves as new viral genomes OR mRNA to be translated into viral proteins and enzymes by

host ribosomes. gag mRNA and pol mRNA are translated into polyproteins that are then cleaved by HIV protease to form capsid proteins and enzymes such as Reverse Transcriptase, Integrase and HIV Protease, respectively. env mRNA is translated into GP160 which is then cleaved into gp120 and gp41 by protease. Maturation: ​gp120 and gp41 is then transported by golgi vesicles to be incorporated into the host cell surface membrane. Capsid proteins formed around viral RNA and enzymes for self-assembly. Nucleocapsid then transported to sites at the host cell surface membrane where gp120 and gp41 have been incorporated into. Release: ​Mature viruses bud off covered with an envelope derived from the host cell surface membrane incorporated with glycoproteins.

Question: Explain the new combination of RNA segments in H7N9. Antigenic shift occurs when 2 or more influenza strains infect the same intermediate host cell simultaneously. The cell replicates the viral genome of the 2 strains at the same time, random assembly of RNA segments from the 3 different influenza viruses- reassortment of NA and HA genes occur leading to new combination of RNA segments in influenza virus when it exits by budding. Question: the need for frequent new flu vaccines. Influenza viruses undergo antigenic drift frequently due to the high mutation rate during its genome replication. Lack of proofreading activity of RNA-dependent RNA polymerase as well as its fast replication involving 2 rounds of complementary RNA strands formed accounts for its high mutation rate. This leads to change in conformation of HA and NA on its viral envelope. Antibodies induced by earlier strains of influenza viruses can no longer bind to the HA since HA is no longer complementary in shape to the antibodies. Hence, new vaccines must be developed frequently.

The viral RNA contains PB2, PB1 and PA which codes for viral RNA polymerase. Explain why such RNA segments are needed by the virus. Viral RNA polymerases required by the viruses would not be present in the host cell. Viral RNA polymerases are able to recognise viral RNA and synthesize complementary RNA strands which serve as template for replication of new viral RNA as well as mRNA for making viral proteins. Explain why HIV protease inhibitors work in treating HIV infection.

HIV proteases cleave HIV polyprotein into several functional proteins such as Reverse Transcriptase, HIV protease and Integrase, required for the formation of mature HIV virions. HIV protease inhibitor is a competitive inhibitor which is complementary to and binds to the active site of the HIV protease, preventing HIV protease from functioning properly so that there is no maturation of HIV virions. This prevents other cells from being infected with HIV and the spread of HIV among human cells. Researchers have recently discovered that the entry of MERS-CoV into its host cells is similar to how Human Immunodeficiency Virus (HIV) enters its host cells. Suggest how MERS-CoV genome enters its host cell. Spike proteins on viral envelope binds to specific receptors on host cell surface membrane. Upon binding, conformational changes occur to the spike proteins that promotes the fusion of the viral envelope and the host cell surface membrane, resulting in the release of MERS-CoV genome into the cytoplasm of the host cell. Researchers predict that MERS-CoV is infectious because as it mutates, it is able to bind to different host cell proteins, leading to viral entry. Suggest why coronaviruses mutate faster than DNA viruses. RNA viruses have higher rates of mutation because the RNA-dependent RNA polymerase/ Reverse transcriptase that synthesizes the RNA genome lacks the proof-reading activity that of DNA polymerases synthesizing DNA viruses’ genomes.