Objectives 22

Define viruses, and identify their typical components (i.e. envelope, Spikes, capsid, etc.).

1) Most simple viruses are nucleic acid (DNA or RNA) wrapped in a protein coat 2) Some also have a lipid membrane: envelope 3) Viruses are NOT cells 4) Infectious agents (possible viral genomes) having either DNA or RNA (never both)•single-stranded or double-stranded 5) Cause infections of humans, animals, plants, and bacteria 6)Cannot carry out any metabolic pathway or reproduce independently, they are olbigate intracellular parasites. Can cause infection in humans, plants, animals, and bacteria. 7) Are VERY small ( usually 20-400 NANOmeters) 8) Hijack the host cell's machinery in order to replicate•No cytoplasmic membrane, cytosol, organelles 9) The protein coat that surrounds the DNA or RNA is called a CAPSID. The capsid is composed of reapeating monomers, protein subunits called capsomeres. Some have an extra layer called an envelope stolen from host cell when released. 10) Enveloped viruses have a lipid membrane surrounding the capsid - envelope, can come from cytoplasmic, nuclear, or golgi membrane. Often has virally encoded glycoprotein spikes important for attachment and release from host cell

Describe the role of adsorption (attachment) in determining host range. In your description, include the terms ligands, receptors, and the role of mutations.

1) Most viruses infect only a particular host, specificity is all about binding, a specif cell type in the host. ---HIV only infects Helper T 2) Bind of viral surface proteins, called ligands, to complementary proteins on surface of host cell receptors. 3) Ex spike protein binding to ACE receptor 1) Most viruses infect only a particular host's cells. 2) Host specificity is due to the binding of viral surface proteins (ligands) to complementary proteins on the surface of the host cell (receptors). 3) Sometimes viruses will only infect a particular type of cell within a particular host. For example, HIV only infects helper T lymphocytes-some people lack a receptor called CCR5 on their T cells (normally HIV binds CCR5). These people are naturally immune to HIV infection!-Some viruses are generalists and can infect many kinds of cells in many hosts (ex. West Nile virus can infect humans, other mammals, birds, and some reptiles) 4) The synthesis of viral genetic material and proteins is DEPENDENT on host cells! Typically viral genomes consist of only a few of the genes needed for synthesis of new viruses.So for a virus to to multiply, it must take over the metabolic machinery of the host cell.•Usually this will destroy the host cell! Viruses that infect bacteria are called bacteriophages

Differentiate the process of release for naked vs. enveloped viruses.

1) Once components of animal viruses are synthesized, they are assembled into virions and are released from the host cell. 2) Enveloped viruses are released via a process called budding, where they acquire a portion of the host cell's membrane that becomes the viral envelope. 3) Budding allows an infected cell to remain alive for some time, it doesnt lyse it. 4) *Virions are extruded through one of the cell's membranes- nuclear, ER, or cytoplasmic* **So the capsid is packaged with the viral genome, then viral glycoproteins are synthesized so when that mRNA is translated it yields gycoproteins. They insert into host membrane. Capsid moves towards it and wraps itself up in host membrane, buds off and goes to infect. 1) Non-enveloped viruses can be released via exocytosis (similar to budding but don't acquire envelope), or they may lyse the cell (similar to bacteriophage release).

Explain the difference between antigenic shift and antigenic drift. Which is more dangerous? Why do you need a flu vaccine every year? How can Influenza move from animals to humans?

Antigenic drift: 1) Accumulation of hemagglutinin and neuriminidase gene mutations within a single strain of virus in a given geographic area. (Basically, a slow change in the antigens on the viral surface over time) 2) The RNA-dependent RNA polymerase does not have a proofreading function. This is crucial to the virus's evolutionary strategy! So it makes mistakes when it makes the complementary strand. Some of these mutations will be beneficiary. 3) If mutations occur in genes that code for hemagglutinin and if you have antibodies that protect you from a particular strain, those antibodies might not be effective for the mutation. Changes in the sequences encoding surface antigens leads to evasion of the immune response! 4) A lot of the mutations wont be infectious but some will be and also evade immune response. *****THIS IS WHY YOU NEED TO GET A VACCINE EVERY YEAR***** Because it happens slowly over time we will have some imunity Antigenic shift: 1) Allows for strains to jump from animals to humans = pandemics 2) Process by which 2 or more different viral strains infect the same host cell, genes are mixed and randomly packaged into virions, and a new virus subtype is formed that has a mixture of surface antigens of the original strains. 2) People will likely have little to no immunity to the new subtype, resulting in severe epidemics or pandemics. 3) When assembly happens, the segments randomly mix re-assort) 4) Only occurs with Influ type A, once a decade •The influenza pandemic of 1918 infected at least 20% of the world's population and killed more people than World War I did (50 million people died during a single flu season).•The 1918 strain arose as a mutant form of an avian influenza strain. Such virulent strains usually arise through antigenic shift, in which genes from two or more different viruses combine.•Humans are generally not infected by avian strains. However, swine can be infected with both avian and mammalian strains and are therefore good "mixing vessels" in which reassortment occurs (see next slide). Influenza•Most recent pandemic: 2009 involved H1N1. •Caused atleast 14,000 deaths worldwide•1918 pandemic was also caused by H1N1•Influenza strains of special concern:•H5N1 kills more than 60% of people who contract the virus from infected birds.•H7N9 affects mainly elderly, killing over 50% of victims. •Resistant to all FDA-approved drugs to treat flu

n detail, contrast the synthesis stage for dsDNA, (+) ssRNA, and (-) ssRNA viruses.

Biosynthesis of ds and ss DNA viruses 1) Viral DNA enters nucleus of host cell and replicates. WIll use host or viral enzymes. Genes transcribed by host RNA polymerase. mRNA exits nucleus and then caposmeres are synthesized and other proteins to build capsid. This happens at the ribosome in the cytoplasm also in nucleus. Capsomeres then go back to nucleus for assembly. They get packaged with that newly copieed DNA = virons. They leave nucleus and go into cytosol and exit cell. Replicate DNA in the nucleus of the host cell using host enzymes or viral enzymes(depending on species). 3) Synthesize capsomeres and other proteins in the cytoplasm using host enzymes•Then capsomere proteins migrate into the nucleus and are joined with newly synthesized DNA to form virions. (so assembly happens in nucleus)•Then virions are released from nucleus into cytosol Biosynthesis of RNA viruses - developed soley in viruses There are 4 types: + ssRNA, -ssRNA, dsRNA, retroviruses* + ssRNA : positive-sense single-stranded RNA. Functions as mRNA 1) The RNA genome can serve directly as mRNA = once genome gets inside the cell it can be translated directly. 2) We need a virally coded RNA polymerase = RNA dependent RNA polymerase that will synthesize a complementary strand off the RNA genome because to need to somehow how the viral genome. So then it can make it and build more genomes. 3) Replication of the RNA genome requires synthesis of a template (-) strand (by a virally coded RNA polymerase). The progeny (+) strands are replicated from the (-) template.*transcription of RNA to RNA it unique to viruses- cells do not do this!* 2) - ssRNA : negative-sense single-stranded RNA. - Doesnt function as mRNA an must make a complement which serves as mRNA. 1) The complement of the genome serves as mRNA. 2) Packages a viral RNA-dependent RNA transcriptase to make (+) strand, which can be translated. Then it uses the (+) strand as a template to make (-) strand to package in virions. Many copies of this will get packaged in the capsid.

What is the difference between (+) ssRNA and (-) ssRNA?

Biosynthesis of ds and ss DNA viruses 1) Viral DNA enters nucleus of host cell and replicates. WIll use host or viral polymerase. Genes transcribed by host RNA polymerase. mRNA exits nucleus and then caposmeres are synthesized and other proteins to build capsid. This happens at the ribosome in the cytoplasm also in nucleus. Capsomeres then go back to nucleus for assembly. They get packaged with that newly copieed DNA = virons. They leave nucleus and go into cytosol and exit cell. Replicate DNA in the nucleus of the host cell using host enzymes or viral enzymes(depending on species). 3) Synthesize capsomeres and other proteins in the cytoplasm using host enzymes•Then capsomere proteins migrate into the nucleus and are joined with newly synthesized DNA to form virions. (so assembly happens in nucleus)•Then virions are released from nucleus into cytosol Biosynthesis of RNA viruses - developed soley in viruses There are 4 types: + ssRNA, -ssRNA, dsRNA, retroviruses* + ssRNA : positive-sense single-stranded RNA. Functions as mRNA 1) The RNA genome can serve directly as mRNA = once genome gets inside the cell it can be translated directly. 2) We need a virally coded RNA polymerase = RNA dependent RNA polymerase that will synthesize a complementary strand off the RNA genome because to need to somehow how the viral genome. So then it can make it and build more genomes. 3) Replication of the RNA genome requires synthesis of a template (-) strand (by a virally coded RNA polymerase). The progeny (+) strands are replicated from the (-) template.*transcription of RNA to RNA it unique to viruses- cells do not do this!* 2) - ssRNA : negative-sense single-stranded RNA. - Doesnt function as mRNA an must make a complement which serves as mRNA. 1) The complement of the genome serves as mRNA. 2) Packages a viral RNA-dependent RNA transcriptase to make (+) strand, which can be translated. Then it uses the (+) strand as a template to make (-) strand to package in virions. Many copies of this will get packaged in the capsid. NOT on EXam dsRNA: double-stranded RNA. (+) strand serves as mRNA. Each strand of the dsRNA acts as template for transcription of its opposite using viral RNA polymerase.Retroviruses: type of +ssRNA virus, but they do not use the (+) strand as mRNA. The (+) strand is reverse-transcribed into dsDNA using an enzyme called reverse transcriptase.*MOST RNA VIRUSES DEVELOP SOLELY IN THE CYTOSOL*.

[Connection - L5] Why do dsDNA viral genomes migrate into the nucleus, but ssRNAgenomes stay in the cytoplasm?

DNA enter the nucleus, RNA have enzymes and stay in cytosol

What is the difference between an enveloped and naked virus? What are the advantages and disadvantages (from a virus' perspective) of each strategy?

Enveloped 1) Enveloped viruses have a lipid membrane surrounding the capsid, called the envelope. 2) The envelope is acquired from the host cell upon release, so it is composed of a phospholipid bilayer and proteins. 3) Some of the proteins are viral glycoproteins (spikes) that play a role in host recognition. 4) Enveloped viruses are easiest to inactivate. Envelope is very fragile and suseptible to damage. This is a downside. But advantage is because envelope is derived from a host cell it will provide some protection from immune system--will recognize as mostly "self". --Spike proteins are virally encoded and can be recognized to IS, but still better protected. NonEnveloped 1) Advange is the capsid is more stable outside the host, protein coat is very tough, protects nucleic acid. It protects the genome from nucleases in biological fluids. - These are enzymes that can chew up nucleaic acid. 2) Downside is more susceptible to recognition by IS, antigens are recognized as foriegn. •Extracellular state•Called virion•Protein coat (capsid) surrounding nucleic acid•Some have phospholipid envelope•Outermost layer provides protection and recognition sites for host cells•Intracellular state•Capsid removed•Virus exists as nucleic acid Glycoproteins - AKA spikes. These are virally encoded. They enable the virus to attach and infect the next host cell.

Big Picture] What types of enzymes must viruses bring (either coded in their geneticmaterial or as proteins) into the cell for synthesis? Why are these enzymes not found inthe host cell?

For RNA viruses, they need their own special RNA polymerases. They have their own enzymes to synthesize complement strands. We dont do RNA to RNA, we do DNA to RNA

[Big Picture] If a mutated papillomavirus (dsDNA) cannot enter the nuclear pore of a hostcell, what stages of the viral life cycle would be impaired, if any? If a mutated filovirus[(-) ssRNA virus] cannot enter the nuclear pore of a host cell, what stages of the viral life cycle would be impaired, if any? Would either of these viruses still be infectious?

If DNA can't enter pore of host, the virus cannot propagate, must get into the nucleus to propagate. RNA dont need to get into nucleus and can still be infectious.

Describe the structure of an Influenza virus (genome, is it enveloped or naked, glycoproteins). What are the H and N spikes? What mechanism does it use to enter and exit host cells? Briefly differentiate between types A,B, and C.

Influenza Epidemiology (FYI)•About 15-20% of the U.S. population gets the flu each year. •36,000 die annually in the U.S.; 250,000-500,000 die annually worldwide. Transmission 1) infection occurs primarily through inhalation of airborne viruses released by coughing or sneezing. -2) Self inoculation- transfer viruses on fingers to nose or mouth 2) Signs and Symptoms• Sore throat, congestion, cough, muscle aches •Sudden fever distinguishes flu from a common cold Process 1) Virus gets in:endocytosis, uncoats, RNA genome inside host cell 2) RNA dependent RNA polymerase that was packaged in the capsid will transcribe mRNA from neg sense (cant be directly translated) must make compliment 3) It will then steal our 5 prime cap off our mRNAs = translation advantage. 4) Once mRNA is made it will serve as template to make more of genomes 5) New copies because each new virus particle has to be packaged with the genome so the special RNA dependent RNA polymerase will read it and then synthesize complementary strands to serve as genomes. Influenza virus STRUCTURE (Orthomyxovirus) Genome 1) ss (-) RNA consists of 8 segments of RNA that make up its genome 2) It is an RNA virus, has its own specific RNA dependent polymerase packaged in the virion 3) It is an Enveloped virus - outer lipid bilayer 4) Glycoproteins spikes - HA (Hemagglutinin), NA (Neuriminidase) 5) Enters cell via ENDOCYTOSIS (membrane wraps around the cell, brings it in, uncoats and end up with RNA genome inside), Exits the cell by BUDDING and how it aquires its envelope --Influenza types A and B cause seasonal epidemics almost every winter in the US. Type C causes a mild respiratory disease and no epidemics, almost all adults have been infected. Lower resp type C are rare. No vaccine for C --Type A can infect animals as well as people. There is concern that changes in type A viruses may cause another major pandemic. B doesnt because it has limited host range (humans and seals) This limits generation of new strains by reassortment. A - can affect many animals and humans, so changes can cause pandemic = means we have little to no immunity Hemagglutinin: (HA) Allows virus to bind to host cells and trigger endocytosis. Facilitates entry of viral genome into host cell.Neuraminidase: (NA) Enables the virus to be released from the host cell. HA and NA are glycoprotein spikes found on the surface of the virus. Oseltamivir (Tamiflu) and Zanamavir are neuraminidase inhibitors. What stage of the replication cycle is affected? The virus will be unable to exit the cell and go infect neighboring cells. FYI: Influenza steals the host cellular 5' mRNA cap!•RNA-dependent RNA polymerase transcribes mRNA from template (-) RNA genome.-Viral mRNA will be capped at 5' end like cellular mRNA-Virus enzyme removes cellular 5' cap, puts it onto ends of viral mRNAs!!- Influenza "pirated cap" provides translation advantage -(+) mRNA serves also as template to make (-) genomes for new virus progeny

Describe the relationship between Chicken pox and Shingles.

Latency example: Chickenpox 1) Caused by a herpesvirus: Varicellovirus (HHV- 2) )•Mode of transmission: inhalation of infected particles from skin lesions 3) Latency: enters the peripheral nerves and moves to a dorsal root ganglion near the spine where it persists as viral DNA indefinitely. 4) Antibodies cannot penetrate into the nerve cell, and because no viral antigens are expressed on the surface of the nerve cell, cytotoxic T cells are not activated. 5) Later in life (due to stress or immunosuppression), the latent virus becomes activated, causing Shingles. Its a different expression of the virus because there is already partial immunity 6) Viruses move along peripheral nerves to the cutaneous sensory nerves of the skin

What does latency mean? What is a provirus? Explain why a virus must be a dsDNA virus(or have the ability to make dsDNA, such as retroviruses) to exhibit latency. How is latency different from lysogeny?

Latent viruses - Animal Viruses 1) Some animal viruses may remain dormant in cells in a process called LATENCY. 2) The latent virus is called a provirus. 3) May be prolonged for years with no signs/symptoms 4) Similar to lysogeny except:***Some latent viruses do not become incorporated into the chromosome. They enter the cell and hang out there. 5) If they do become integrated, they cannot be excised (i.e. the provirus never comes out of the chromosome). So induction does not occur. Even if it remains though, all the genes can be expressed. The viruses can get synthesized, and can get an active infection. EX HIV

Diagram the lytic and lysogenic cycle of viruses. If a virus is in a lysogenic state, is the stage permanent? Explain why or why not.

Life Cyles of Bacteriophage Lytic - Like an active infection. Virus gets in and takes control over cell, reproduce, blow up, and infect neighboring cells. 1 ): Attachment (Adsorption)- Proteins on the virus recognize receptor proteins on the surface of the host's cell wall. The virus attaches to the host cell using tail fibers 2) : Entry- the virus releases lysozyme to degrade the peptidoglycan of the cell wall to facilitate entry. The phage's tail contracts, forcing a hollow tube through the cell wall/membrane. The virus injects its genome through the tube. Viral enzymes (either carried in the capsid or coded by viral genes and made by the bacterium) degrade bacterial DNA. 3): Synthesis: New viruses are synthesized-- Viral nucleic acid is transcribed and translatedby host ribo by the bacterial host, meaning nucleic acid is replicated. Copies need to be made each new phage will need to be packaged with a copy of the genome. Resulting in viral nucleic acid and protein that will be assembled into new virus particles. 4): Assembly: Viral nucleic acid is packaged into capsids. Sometimes, host DNA is "accidentally" packaged into capsids... now when the virus infects a new host, it will inject the old host's DNA into the new host (transduction, lecture 11) 5) Release- Newly assembled viruses are released, and the cell lyses open. *LYSOGENIC cycle of bacteriophages - like latent/dormant stage that phage can enter. Only some phage can do this cycle, some can do both, 1) Viral DNA enters the cell as in the lytic cycle, but the host cell's DNA is not destroyed. The phage's genome does not immediately assume control of the cell. 2) Viral DNA is incorporated into the host chromosome, and remains inactive and is called a prophage, until conditions for viral replication are favorable. 3) The host cell resumes normal function, but when binary fission happens, it replicates its genome, the viral DNA will be replicated as well. Daughter cells will acquire the prophage. Prophage may remain part of chromosomes for generations or forever. 4) Eventually, the prophage may be excised from the chromosome (called induction), and now the lytic cycle can begin. Why go into lysogenic cycle? If there are no bacteria around its able to infect. Quorum sensing signals cause prophage induction. Or also low levels of antibiotics or UV light because of the potential threats to the cell because then the virus dies. Why should we care? Bacterophage Therap, using bacteriaphage to kill bacteria. Even our microbiome has bacteriophage. In our gut, enteriococci house lysogenic phage and if there are potential pathogens competing...induction will happen and bacteriophage will be synthesized and released and infect and

What are three mechanisms of entry of animal viruses?

Replication of Animal Viruses - these include viruses that infect humans but there are a lot of similarities with lytic and lysogenic cycle. 1) Attachment of an animal virus depends on binding of virion proteins to protein/glycoprotein receptors in animal cell's cytoplasmic membrane. With the bacteriophage, it could be binding to some part of the cell wall or recep on fimbria. 2) Animal viruses lack tails and tail fibers- they have glycoprotein spikes or other attachment molecules on their capsids or envelopes. 3) Viruses enter host cell via one of the mechanisms A) Direct penetration - this would be with a non enveloped virus. So viral genome will bind to some receptors on cyto membrane, capsid just sits therem the genome itself enters. Takeway is all 3 mechanisms you end up with just viral genome inside cell B) Membrane fusion - happens with enveloped viruses, it binds to receptors on cyto membrane and because envelope derived from phospho billayer and binds to it, it kinds fuses together and then the capsid enters the cell and falls apart, it uncoats and you end up with viral genome inside. Because they fused, you will have viral glycoproteins stciking out of the side of plasma membrane C) Endocytosis - You have binding of enveloped virus and its engulfed and envelope comes apart and capsid too and again you end up with viral genome but this time there are no glycoproteins remaining in the cyto membrane.

What is avian (bird) flu? What is the human mortality rate of avian flu? Despite the current situation where avian flu is not easily transmitted from birdto human, there is great concern that avian flu could cause a pandemic. Why is thisconcern a valid one?

•H5N1 kills more than 60% of people who contract the virus from infected birds.•H7N9 affects mainly elderly, killing over 50% of victims. •Resistant to all FDA-approved drugs to treat flu •Avian strains currently have limited tropism for the human respiratory tract, and none are readily transmitted between humans (but this could change as a result of mutation)! Right now its not easily transmissible but it could eventually be

[Connection - L17] Cells infected with latent or lysogenic viruses may exhibit new properties. What is one example of a disease that we discussed in class caused by a bacterium infected with a lysogenic phage? What new property does the virus confer to the host cell?

•One result of lysogeny is the host cell may exhibit new properties. It can encode certain virulence factors•E coli 0157:h7: the gene that codes for Shiga toxin is a prophage gene!•Only Streptococci carrying a lysogenic phage are capable of causing toxic shock syndrome. •Another result is Specialized Transduction. •Remember Generalized Transduction? ANY bacterial gene could be packaged in a phage capsid.• In Specialized Transduction, only certain genes can be transferred. Lysogenic phage will package its own DNA with Bacterial DNA in same capsid. When a phage is excised , adjacent genes from either side may remain attached to phage DNA and that can be packaged into transducing phage.

[Connection - L7] Explain, in detail, the 2 mechanisms of transduction. Identify the role ofthe lysogenic and lytic cycle in each.

•One result of lysogeny is the host cell may exhibit new properties. •E coli 0157:h7: the gene that codes for Shiga toxin is a prophage gene!•Only Streptococci carrying a lysogenic phage are capable of causing toxic shock syndrome. •Another result is Specialized Transduction. •Remember Generalized Transduction? ANY bacterial gene could be packaged in a phage capsid.• In Specialized Transduction, only certain genes can be transferred. Review: Transduction(horizontal gene transfer via a bacteriophage) In generalized transduction (shown here), all genes contained within a bacterium infected by a phage are equally likely to be packagedin a phage coat and transferred. In specialized transduction, (next slide) only certain bacterial genes are transferred Specialized Lysogenic phage packages bacterial DNA along with its own DNA in the same capsid. •When a prophage is excised from the host chromosome, adjacent genes from either side may remain attached to the phage DNA.