Fucking Exam 3

What are viruses made of? What does it mean that viruses are “obligatory intracellular parasites”? What is a mature virus particle called?

• Viruses are composed of protein and nucleic acid. Some viruses also have a lipid envelope.

• Viruses cannot replicate on their own. They must infect a host cell and use host-cell machinery to make more virus.

• Mature virus particle = A virion

What is the capsid? What are bacteriophages?

A capsid is the protein shell of a virion that surrounds/protects the viral nucleic acid.

Bacteriophages are viruses that infect bacteria.

Give examples of viruses and the diseases they cause.

Poliovirus → poliomyelitis; poxvirus → smallpox; herpesviruses → cold sores/genital herpes; hepatitis viruses → hepatitis; Ebola virus → Ebola hemorrhagic fever; HIV-1 → AIDS; coronavirus → COVID-19. Flaviviruses include yellow fever virus and Zika virus: yellow fever virus causes yellow fever, while Zika virus is associated with microcephaly.

What are the two major ways viruses cause disease?

1. Cytopathic effect, where viral replication damages or kills infected cells. 2. Host immune responses, where immune-mediated destruction or inflammation damages tissue.

What is cytopathic effect, or CPE?

CPE is visible damage to infected cells caused by viral replication. Cells may become sick, die, detach, fuse, or form abnormal structures.

What is a syncytium?

A multinucleated giant cell formed when infected cells fuse together. It is one type of viral cytopathic effect.

Can viruses cause disease even if they do not directly kill cells?

Yes. Some viruses do not cause strong CPE, but the immune system recognizes infected cells and destroys them, causing tissue damage.

How can SARS-CoV-2 cause acute respiratory distress syndrome, or ARDS?

By a direct effect, causing CPE in pulmonary epithelial cells, and an indirect effect, causing immune-cell infiltration and release of inflammatory cytokines such as TNF-α and IL-6, producing a cytokine storm.

What is a cytokine storm?

An excessive inflammatory response where immune cells release large amounts of cytokines, such as TNF-α and IL-6, causing tissue damage.

How can viruses contribute to cancer? Approximately what percentage of human cancers are caused by viruses?

Viral infection can cause abnormal cell growth, sensitize cells to mutations, cause chronic inflammation, damage tissue, and promote cellular transformation.

Approximately 20% of human cancers are caused by viruses.

Which virus is associated with cervical carcinoma? Which viruses are associated with hepatocellular carcinoma?

Cervical carcinoma = Human papillomavirus, or HPV.

Hepatocellular carcinoma = Hepatitis B virus and hepatitis C virus.

Why is it difficult to treat viral diseases with antiviral drugs? What is the best way to control viral diseases?

Antiviral drug development often takes 10 or more years, and many antivirals are only partially effective.

Vaccination is the best way to control viral diseases.

Why are vaccines emphasized over antivirals

Vaccines can prevent infection or severe disease before widespread viral replication occurs, whereas antivirals are difficult to develop and may only be partially effective.

Vaccines are given before infection to prime the immune system and help prevent disease before it appears. (Prophylactic)

Vaccines can provide long-term immune memory and have been used to eradicate some viral diseases.

What is a plaque assay used for? What is a plaque? What is the general formula for calculating viral titer from a plaque assay?

A plaque assay is used to measure the amount of infectious virus in a sample.

A plaque is a clear zone or area of dead/damaged cells caused by infection and spread of virus from an initially infected cell.

PFU/mL = number of plaques ÷ (dilution × volume added in mL).

What does PFU stand for? What does PFU measure? Why is PFU not always equal to the total number of viral particles?

• PFU = Plaque Forming Unit.

• PFU measures the number of infectious viral particles capable of forming plaques.

• Not all viral particles are infectious. The lecture example states that only about 1 in 10,000 HPV particles is infectious.

What does MOI stand for? What is MOI? What is the formula for MOI?

• MOI = Multiplicity of Infection.

• MOI is the number of PFU per cell.

• MOI = PFU ÷ number of cells.

• We use Poisson distribution for MOI because viral infection of cells is probabilistic; the Poisson distribution estimates the probability that cells receive 0, 1, or more infectious particles

If you infect 1 million cells with 1 million PFU, what is the MOI?

MOI = 1,000,000 PFU ÷ 1,000,000 cells = 1.

Does an MOI of 1 mean every cell is infected?

No. Because infection follows a Poisson distribution, an MOI of 1 infects about 63.2% of cells, not 100%.

What equation is used to calculate the probability that cells are not infected?

P(0) = e⁻ᵐ, where P(0) is the probability of no infection and m is the MOI.

In the Poisson equation P(0) = e⁻ᵐ, what does P(0) mean? In the Poisson equation P(0) = e⁻ᵐ, what does m mean?

P(0) is the probability that a cell receives zero infectious viral particles, meaning the cell is not infected.

m is the multiplicity of infection, or MOI.

What percentage of cells are infected at MOI = 1?

P(0) = e⁻¹ = 0.368, so 36.8% are uninfected. Therefore, 63.2% are infected.

What MOI is needed for 99% infection? How many PFU are needed to infect 1 million cells at 99% infection?

For 99% infection, P(0) = 0.01. m = −ln(0.01) = 4.6.

MOI needed = 4.6. PFU = 4.6 × 1,000,000 cells = 4.6 × 10⁶ PFU.

17 plaques were seen at a 10⁻⁶ dilution using 0.1 mL. What is the viral titer?

PFU/mL = 17 ÷ (10⁻⁶ × 0.1) = 17 ÷ 10⁻⁷ = 1.7 × 10⁸ PFU/mL.

What does ELISA detect in virology? What does PCR detect in virology? What can electron microscopy be used for in virology?

• ELISA is a serological method that can detect viral antigens or antiviral antibodies.

• PCR detects viral genetic material using viral genome-specific primers.

• Electron microscopy can visualize viral particles, often using negative staining.

What are the two major geometric types of virion shells?

1. Helical symmetry.

2. Cubic symmetry, usually based on an icosahedron.

What is helical symmetry? What is an example of a virus with helical symmetry?

• Protein subunits are arranged in a helix around the viral genome

• Example: Tobacco mosaic virus.

What is cubic symmetry in viruses?

A near-spherical, closed-shell arrangement usually based on an icosahedron.

What is an icosahedron? Why is the icosahedron important in virology?

• A geometric structure with 20 equilateral triangular faces and 12 vertices.

• Many viruses use icosahedral symmetry to build efficient capsids from repeated protein subunits.

What is a pentamer? Where are pentamers found in an icosahedral capsid?

• A capsomer made of 5 protein subunits.

• At the 12 vertices of the icosahedron.

What is a hexamer?

A capsomer made of 6 protein subunits.

What is the formula for total number of capsomers in an icosahedral shell?

• C = (T − 1) × 10 + 12, where C is capsomers and T is the triangulation number.

• T is the triangulation number, or number of subtriangles.

If a virus has T = 1, how many capsomers does it have? If a virus has T = 4, how many capsomers does it have?

T=1: C = (1 − 1) × 10 + 12 = 12 capsomers.

T=4: C = (4 − 1) × 10 + 12 = 30 + 12 = 42 capsomers.

For hepatitis B virus with T = 4, how many pentamers and hexamers are present? How many total protein subunits are present?

• 12 pentamers and 30 hexamers.

• 12 pentamers × 5 = 60 subunits; 30 hexamers × 6 = 180 subunits; total = 240 protein subunits.

What is the difference between a naked virus and an enveloped virus?

A naked virus lacks a lipid envelope. An enveloped virus has a lipid envelope surrounding the capsid/nucleocapsid.

Can enveloped viruses have icosahedral capsids? Can enveloped viruses have helical capsids? What is adenovirus used to demonstrate?

• Yes. Some enveloped viruses contain an icosahedral capsid inside the envelope.

• Yes. SARS-CoV is shown as an example of an enveloped virus with a helical capsid.

• Adenovirus is shown by electron microscopy as an example of an icosahedral virus structure.

Give an example of a naked ssDNA, naked dsDNA virus families, naked dsRNA virus family, enveloped ssRNA virus families, and enveloped dsDNA virus families

• Naked ssDNA virus family = Parvovirus.

• Naked dsDNA virus families = Papillomavirus and adenovirus.

• Naked dsRNA virus family = Reovirus.

• Enveloped ssRNA virus families = Togavirus, retrovirus, coronavirus, paramyxovirus, rhabdovirus, and orthomyxovirus.

• Enveloped dsDNA virus families = Herpesvirus and poxvirus.

What are the steps of the viral life cycle?

1. Adsorption. 2. Penetration. 3. Uncoating. 4. Eclipse. 5. Maturation.

• Adsorption = The attachment of virions to surface receptors on target cells. This helps determine tissue tropism, meaning which tissues the virus can infect. Adsorption is an electrostatic interaction. High-salt or low-pH solutions can interrupt adsorption.

• Penetration = The process by which the virus or viral genome enters the host cell. This helps determine tissue tropism, meaning which tissues the virus can infect.Many bacteriophages inject their genome into the bacterial host cell. Receptor binding may trigger receptor-mediated endocytosis, allowing the virus to enter the cell. The virus enters the cell by mechanisms such as receptor-mediated endocytosis, membrane fusion for many enveloped viruses, or direct genome injection in bacteriophages.

• Uncoating = Uncoating releases the viral genome from the capsid. It may occur at the plasma membrane, in the cytoplasm, or in the nucleus. The viral capsid/exterior shell is removed, releasing the viral genome into the cell.

• Eclipse = The eclipse phase is the early stage when viral infectivity disappears because the virus has uncoated and the viral genome is directing replication events. Called eclipse because infectious virions temporarily disappear because the incoming virus has uncoated, but new mature infectious virions have not yet been produced. In the eclipse phase the uncoated viral genome directs biochemical events needed for viral replication, including viral mRNA production, protein synthesis, and genome replication. Infectious virus temporarily “disappears” because the virion has disassembled, but new infectious virions have not yet formed. During this phase, the viral genome directs mRNA production, viral protein synthesis, and genome replication.

• Maturation = New viral genomes are packaged by capsid proteins to form nucleocapsids. Structural proteins include capsid, envelope, and matrix proteins; nonstructural proteins include enzymes/regulatory proteins used for replication. Release: viruses exit by lysis, where the cell bursts and dies, or by budding, where enveloped viruses acquire a lipid envelope from a cellular membrane and the host cell may survive for some time.

What determines tissue tropism? What is tissue tropism? What is the tissue tropism of poliovirus? What is the tissue tropism of hepatitis A virus?

• Tissue tropism is determined largely by whether the target tissue expresses the correct viral receptor.

• Tissue tropism means a virus can infect only certain tissue types because its receptor is found only on certain cells.

• Poliovirus can infect intestinal tissues and the spinal cord, but not all tissues.

• Hepatitis A virus infects intestinal tissues and the liver.

What do viral mRNAs encode?

Viral mRNAs encode non-structural proteins and structural proteins.

What are non-structural viral proteins? What are structural viral proteins?

• Non-structural viral proteins = Proteins that are not part of the final virion structure. They may be enzymes for genome replication, enzymes for protein maturation, or regulatory proteins.

• Structural viral proteins = Proteins that become part of the virion, such as capsid proteins, envelope proteins, and matrix proteins.

What happens after non-structural proteins such as viral RNA polymerase are made? Do all DNA viruses encode their own DNA polymerase? (in viral life cycle)

The viral genome is replicated to make progeny viral genomes.

No. Some DNA viruses produce their own DNA polymerase, while others use the host cell’s DNA polymerase.

What happens during nucleocapsid formation? (in viral life cycle)

Capsid proteins package progeny viral genomes to form the nucleocapsid.

How many progeny genomes can one incoming viral genome produce?

One copy of an incoming viral genome can direct production of tens of thousands of progeny viral genomes.

How do enveloped viruses mature? How do some enveloped viruses acquire their envelope?

• The nucleocapsid interacts with envelope proteins and often matrix proteins to form the mature enveloped virus.

• Some acquire their envelope from the plasma membrane as they bud from the cell surface.

What are two general patterns of viral release?

Some viruses are released in bulk when the cell dies. Others are released individually before the cell dies, often by budding.

Why do different viral genome types have different replication strategies?

All viruses must ultimately produce mRNA to make proteins, but they start from different genome types: dsDNA, ssDNA, dsRNA, positive-sense RNA, negative-sense RNA, retroviral RNA, or hepadnaviral DNA/RNA intermediates.

What is the key point for dsDNA viruses? What is the key point for ssDNA viruses? What is the key point for dsRNA viruses? What is the key point for positive-sense ssRNA viruses? What is the key point for negative-sense ssRNA viruses? What is the key point for retroviruses?

• dsDNA viruses can produce mRNA, which is translated into proteins.

• ssDNA viruses first form dsDNA, then produce mRNA and proteins.

• dsRNA viruses produce mRNA, which is translated into proteins.

• Positive-sense ssRNA can function as mRNA, but replication involves making a negative-strand intermediate.

• Negative-sense ssRNA must first be copied into mRNA before proteins can be made.

• Retroviruses convert ssRNA into DNA using reverse transcription, then form dsDNA, mRNA, and proteins.

Different genome types use different routes, but the central goal is the same: make viral mRNA, viral proteins, and progeny genomes.

What is viral pathogenesis? What are the three major steps of viral pathogenesis?

Viral pathogenesis is the process by which viruses cause disease

1. Entry of the virus into the host.

2. Local replication at the entry site.

3. Dissemination to target organs where disease occurs.

What are major routes of viral entry into the host? Which route is the most common way of viral entry?

• Alimentary tract (poliovirus and norovirus) , respiratory tract (rhinovirus and influenza virus) , urogenital tract (HIV-1 and herpes simplex virus-2) , and skin (abies virus and papillomavirus) .

The respiratory tract is the most common route of viral entry.

What is viral dissemination? What is the most common route of viral dissemination?

Viral dissemination is the spread of virus from the entry site to other tissues.

Most common route = Through the circulatory system

What is the pathogenesis sequence of poliovirus? Why can poliovirus cause paralysis?

Ingestion of contaminated food → infection of intestinal epithelial cells → release into lymphatic system → bloodstream/viremia → infection of CNS → paralysis.

After intestinal replication and dissemination through blood, poliovirus can infect the central nervous system, leading to paralysis.

What are arboviruses? Give examples of arboviruses. What is the transmission pathway of arboviruses? Why are salivary glands important in arbovirus transmission?

• Arboviruses are arthropod-borne viruses transmitted by blood-sucking insects such as mosquitoes, fleas, and ticks.

• Examples: West Nile virus, yellow fever virus, dengue virus, and Zika virus.

• Patient → blood-sucking insect takes blood meal → virus enters insect midgut → infects epithelial cells → viremia in insect → infects salivary gland → virus enters saliva → transmission to another patient.

• The virus must infect the insect salivary glands so it can be transmitted through saliva during the next bite.

What are PRRs? What do PRRs recognize? What can PRR activation lead to?

• Pattern-recognition receptors. They detect signs of infection or damage and trigger antiviral responses.

• PRRs recognize PAMPs and DAMPs.

• PRR activation can lead to cytokine expression, including interferons and TNF-α, induction of apoptosis, and induction of inflammasomes.

What are PAMPs? What are examples of viral PAMPs?

• Pathogen-associated molecular patterns, which are molecular features associated with pathogens.

• Examples: 5′-triphosphate RNA, double-stranded RNA, polyU sequence, and cytoplasmic double-stranded DNA.

What are DAMPs? What are examples of DAMPs?

• Damage-associated molecular patterns, which are host molecules released or exposed during cell stress or damage.

• Examples: Extracellular ATP, HMGB1, heat shock proteins, and F-actin.

What are RIG-I-like receptors? What do RIG-I-like receptors recognize?

• Cytoplasmic PRRs including RIG-I, MDA5, and LGP5 that recognize viral RNA patterns.

• They recognize 5′-triphosphate RNA, ssRNA such as polyU sequence, and dsRNA

What do Toll-like receptors recognize? How many TLRs do humans have?

• TLRs recognize RNA, DNA, peptidoglycans, and other molecules either on the cell surface or in endosomes.

• Humans have 10 TLRs.

What are NOD-like receptors, or NLRs? What are the three NLR subfamilies?

• Intracellular PRRs activated by virus-associated PAMPs and DAMPs. They can also be activated by TLR signaling

• NODs, NLRPs, and IPAF

What does cGAS recognize? What is the cGAS-STING pathway?

• cGAS recognizes naked double-stranded DNA in the cytoplasm

• cGAS → cGAMP → STING → TBK → IRF3 → IFN-β

Why is interferon important in antiviral defense? What is apoptosis in the context of antiviral defense? What are inflammasomes in antiviral defense?

• Interferon helps induce an antiviral state and coordinates immune responses that limit viral replication

• Programmed cell death that can eliminate infected cells before they produce more virus

• Inflammatory signaling complexes that can promote cytokine release and inflammatory responses during infection

What is autophagy? Why is autophagy important for normal cells? How can autophagy help control viral infection?

• Autophagy is a cellular recycling process that removes protein aggregates, damaged organelles, and intracellular pathogens

• It maintains cellular homeostasis by degrading and recycling damaged components such as ribosomes and mitochondria

• Cells can use autophagy to capture and degrade intracellular pathogens, including viruses

What are the major structures in macroautophagy? What is a phagophore? What is an autophagosome? What is an autolysosome?

• Macroautophagy: Phagophore → autophagosome → autolysosome after fusion with a lysosome

• Phagophore: The initial membrane structure that begins surrounding material targeted for autophagy

• Autophagosome: A vesicle that encloses material targeted for degradation, such as damaged organelles or pathogens

• Autolysosome: The structure formed when an autophagosome fuses with a lysosome, allowing contents to be degraded

Why can positive-sense ssRNA act quickly after infection?

Positive-sense ssRNA can function like mRNA, so it is directly ready for translation into viral proteins.

What must negative-sense ssRNA viruses do before making proteins?

They must first make positive-sense mRNA using a viral RNA polymerase.

What does it mean for an RNA virus to be positive-stranded? hat does it mean for an RNA virus to be negative-stranded? Give examples of viruses

• A positive-stranded RNA genome can function like mRNA after entry into the cell, so it can be directly translated by host ribosomes

  • Example: Poliovirus, a picornavirus.

• A negative-stranded RNA genome is complementary to mRNA, so it must first be copied into positive-sense mRNA by a viral RNA polymerase/transcriptase before proteins can be made.

  • Example: Influenza virus, an orthomyxovirus.

What are picornaviruses?

Picornaviruses are small positive-stranded RNA viruses. Examples include poliovirus, rhinovirus, foot-and-mouth disease virus, hepatitis A virus, and human parechovirus.

What type of genome does poliovirus have? What is the importance of the picornavirus 5′ UTR? What is VPg? What happens to VPg during poliovirus eclipse, and what is VPg used for later?

• Picornaviruses are small positive-stranded RNA viruses. Examples include poliovirus, rhinovirus, foot-and-mouth disease virus, hepatitis A virus, and human parechovirus.

• The 5′ UTR contains highly conserved secondary structures that form an IRES, or internal ribosomal entry site, which allows viral translation to begin internally.

• VPg is a small viral protein covalently attached to the 5′ end of the picornavirus genome. It also functions as a protein primer during poliovirus RNA synthesis.

• During eclipse, VPg is removed/delinked from the viral RNA so the RNA can associate with ribosomes. Later, VPg functions as a protein primer for poliovirus RNA replication.

What does IRES stand for, and why is it important?

IRES = internal ribosomal entry site. It lets picornavirus RNA initiate translation without needing the normal 5′ cap-dependent translation mechanism.

Picornavirus RNA lacks a normal 5′ cap, so it cannot rely on standard cap-dependent translation. The IRES recruits ribosomes internally and allows viral protein translation to occur despite missing a 5′ cap.

Why can picornaviruses translate their RNA even when host cap-dependent translation is blocked? How fast is the picornavirus replication cycle? Most picornaviruses cause what type of infection?

• Picornaviruses use an IRES-mediated, cap-independent translation mechanism, so they do not depend on intact host cap-binding translation machinery.

• The picornavirus replication cycle is completed approximately 5–10 hours after infection

• Most picornaviruses cause lytic infection, meaning infected cells eventually lyse and release progeny viruses. Hepatitis A virus is noted as an exception.

What receptor does poliovirus use for adsorption? What is adsorption in the poliovirus life cycle?

• Poliovirus uses CD155, a member of the immunoglobulin superfamily.

• Adsorption is the attachment of the virus to a specific receptor on the host cell surface.

What happens after poliovirus binds its receptor?

Receptor binding triggers receptor-mediated endocytosis and causes structural changes in the virion, including VP1 conformational change and loss of VP4.

What is the role of VP1 during poliovirus entry? What happens to VP4 during poliovirus entry?

• After receptor binding, VP1 changes conformation and exposes a hydrophobic domain that helps the virus interact with the membrane during entry/uncoating.

• VP4 is lost after receptor binding, helping prepare the virion for uncoating

Where can poliovirus uncoating occur? What happens during the eclipse phase of poliovirus infection?

• Poliovirus uncoating may occur at the cell surface or on endosomal membranes.

• During eclipse phase, he viral genomic RNA is released into the cytosol, VPg is removed, and the RNA associates with ribosomes for protein synthesis

Where does poliovirus replication and assembly occur? What is the first major protein product made from poliovirus RNA?

• Poliovirus replication and assembly occur entirely in the cytoplasm.

• Poliovirus RNA is translated into one large polyprotein, which is then cleaved into functional viral proteins.

Which poliovirus proteins are proteases and what do they do?

• 2A is a protease, and 3C is the major viral protease.

• 2A cleaves the P1/P2 junction of the viral polyprotein.

• 3C cleaves the P2/P3 junction and, along with precursor 3CD, cleaves many other viral polyprotein junctions.

What is the function of poliovirus 3D? What is the function of poliovirus 2B? What is the function of poliovirus 3B? 3B is VPg, the viral genome-linked protein and protein primer.

• 3D is the viral RNA-dependent RNA polymerase.

• 2B increases membrane permeability, alters membrane structure, and may help with virus release and cell death.

What is the basic idea of poliovirus RNA replication? What is the basic maturation sequence for poliovirus assembly? How is poliovirus released from infected cells?

• The positive-strand RNA is copied into a negative-strand template, which is then used to make many new positive-strand genomes.

• VP0, VP1, and VP3 form a protomer → five protomers form a pentamer/capsomer → twelve pentamers form a procapsid → genomic RNA is packaged → VP0 is cleaved into VP2 and VP4 → mature virion.

• Poliovirus matures inside the cell and remains there until the cell disintegrates/lyses, releasing progeny virions.

What is eIF4F? What does eIF4E do in cap-dependent translation? What does eIF4G do in cap-dependent translation? Why does cleavage of eIF4G not stop poliovirus protein synthesis? (Poliovirus)

• eIF4F is a host translation initiation complex made of eIF4G, eIF4E, and eIF4A.

• eIF4E binds the 5′ cap of host mRNAs.

• eIF4G helps connect the capped mRNA to the 40S ribosomal subunit through interactions with other initiation factors.

• Poliovirus translation is IRES-mediated and cap-independent, so it does not require intact eIF4G in the same way host cap-dependent mRNAs do.

How does poliovirus shut off host protein synthesis?

Poliovirus 2A protease cleaves eIF4G, disrupting the eIF4F initiation complex and blocking host cap-dependent translation.

How can poliovirus harm the host cell while continuing to make its own proteins?

Poliovirus uses 2A protease to cleave eIF4G, shutting off host cap-dependent translation, while its own RNA uses an IRES to continue viral protein synthesis.

What is a neutralizing antibody? How can neutralizing antibodies block viral infection at the adsorption step? For influenza, what viral protein is especially important for neutralizing antibody binding? What is the relationship between antigenic drift and neutralizing antibodies?

• A neutralizing antibody binds a virus in a way that prevents infection, often by blocking receptor binding, entry, or fusion.

• They can bind viral surface proteins near the receptor-binding region, preventing the virus from attaching to its host cell receptor.

• HA, because HA mediates receptor binding and entry, and the HA trimer contains receptor-binding and neutralizing antibody-binding regions.

• Antigenic drift produces mutations in antibody-targeted epitopes, allowing influenza mutants to escape neutralizing antibodies.

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What does HA stand for, and what does it do? What does NA stand for, and what does it do? (Influenza)

• HA = hemagglutinin. It binds the host cell receptor and mediates membrane fusion during entry.

• NA = neuraminidase. It removes neuraminic acid/sialic acid and helps newly formed virions disperse rather than aggregate.

What does M1 do in influenza virus? What does M2 do in influenza virus?

• M1 is the matrix protein. It underlies the viral envelope and interacts with HA, NA, M2, and RNP.

• M2 is an ion channel that allows protons into the virion during entry, helping uncoating.

What proteins make up the influenza transcriptase complex? What are the function of each proteins

• PB1, PB2, and PA form the transcriptase complex.

• PB1 performs nucleotide addition during RNA synthesis.

• PB2 binds the 5′ cap of host RNA during cap-snatching.

• PA has endonuclease activity and cleaves host capped RNA during cap-snatching.

What receptor does influenza virus bind? and why is this linkages important for influenza host range?

• Influenza HA binds N-acetylneuraminic acid, also called sialic acid.

• Different sialic acid linkages to galactose influence which host species a particular influenza virus can infect.

What is the first step of influenza virus entry?

• HA binds sialic acid on the host cell surface.

• Binding triggers endocytosis, bringing the virus into an endosomal compartment.

• Low pH in late endosomes/lysosomes causes HA to change conformation, exposing the fusion peptide.

What does the HA fusion peptide do? What is the final result of HA-mediated membrane fusion?

• The fusion peptide inserts into the endosomal membrane and helps fuse the viral envelope with the endosomal membrane.

• Formation of a fusion pore that allows viral contents/RNPs to enter the cytoplasm.

What role does M2 play during influenza uncoating? Where do influenza RNPs go after uncoating?

• M2 allows protons from the endosome to enter the virion, causing dissociation of M1 from HA, lipid, and RNP, which helps release RNPs.

• he RNPs enter the nucleus through nuclear pores.

Why must influenza carry its own RNA transcriptase complex?

Its genome is negative-sense RNA, so it cannot be directly translated. It must first be transcribed into mRNA by viral polymerase.

What is primary transcription in influenza infection? What is cap-snatching? Which influenza proteins are involved in cap-snatching?

• Primary transcription is the first synthesis of viral mRNA from the negative-sense virion RNA genome.

• Cap-snatching is when influenza polymerase steals capped fragments from host RNAs to use as primers for viral mRNA synthesis.

• Influenza viral mRNAs need a 5′ cap for translation. The viral polymerase steals capped fragments from host RNAs and uses them as primers to make viral mRNAs.

• PB2 binds the cap, PA cleaves the host RNA, and PB1 extends the viral RNA chain.

Why is influenza mRNA not a complete copy of the viral RNA template?

Viral mRNA synthesis stops before the end of the negative-strand template, and poly(A) tails are added at oligo(U) sequences.

What is the basic concept of influenza RNA replication?

Negative-strand virion RNA is copied into positive-sense antigenomic RNA, which then serves as a template to make new negative-strand virion RNA.

What is the antigenome in influenza replication?

The antigenome is the positive-sense RNA template used to make new negative-strand virion RNA genomes.

What protein helps shut off synthesis of positive-stranded influenza RNA later in infection? What does NS2 do in influenza infection?

• M1 helps shut off synthesis of positive-stranded RNA, including template RNA and mRNA, later in infection.

• NS2 contains a nuclear export signal and is required for export of viral RNPs from the nucleus.

What is the function of influenza NA during release? What happens if influenza NA is defective?

• NA digests neuraminic acid/sialic acid, helping new virions separate from the cell and from each other.

• If defective, virions can aggregate because they are not efficiently released/dispersed.

What are the two influenza RNA segments that undergo splicing? and what genes are encoded + functions

• RNA segment 7 and RNA segment 8.

• Segment 7 encodes M1 and M2.

• Segment 8 encodes NS1 and NS2.

  • NS1 helps influenza evade host defenses by limiting IFN-β induction, inhibiting antiviral PKR and OAS/RNase L pathways, blocking cellular mRNA processing/export, enhancing viral mRNA translation, and helping prevent premature cell death.

Why do some influenza drugs target M2? Why does inhibiting M2 suppress influenza infection? Why does inhibiting NA suppress influenza spread?

• Targeting M2 to suppresses uncoating.

• M2 is needed for proton entry into the virion and uncoating, so blocking M2 interferes with release of RNPs.

• NA helps release and disperse newly formed virions, so NA inhibition limits viral spread.

Why can influenza undergo genetic reassortment? What is genetic reassortment? Why is genetic reassortment important for influenza pandemics?

• Influenza has a segmented genome with 8 separate RNA segments, so if two different influenza viruses infect the same cell, their genome segments can mix.

• Genetic reassortment is the mixing of genome segments from two different influenza viruses infecting the same cell, producing progeny viruses with new segment combinations.

• Reassortment can generate a virus with a new HA, and sometimes a new NA, that humans have little preexisting immunity against, allowing widespread infection.