L3 - Picornaviruses I (Andrew Davidson)

Question 1

What is the Picornaviridae family

Answer 1

a large family of small, non enveloped +ve sense RNA viruses (Pico = small, rna = RNA, viridae = virus)

Question 2

how many species and genera of picornaviridae are there

Answer 2

147 species divided across 63 genera

Question 3

What are some of the major genera within the picornaviridae family?

Answer 3

include Enteroviruses (e.g. poliovirus), Rhinoviruses and Hepatoviruses (Hep A virus)

Question 4

How diverse is the rhinovirus species group within picornaviruses?

Answer 4

Rhinoviruses are highly diverse, with ~160 known serotypes, all classified under a single species within the picornavirus group

Question 5

why is picornaviridae medically significant?

Answer 5

includes several important human pathogens, such as polioviruses, hepatitis A virus, rhinoviruses, and foot-and-mouth disease virus that play significant roles in causing diseases ranging from the common cold to serious conditions like poliomyelitis and hepatitis A.

Question 6

Which virus in the Picornaviridae family is nearly eradicated, and why is it notable?

Answer 6

Poliovirus is nearly eradicated and is notable for being extensively studied due to the disease it causes, poliomyelitis. Its importance led to major research efforts beginning in the 1950s, making it the first eukaryotic virus to be grown in cultured cells . . This allowed scientists to conduct detailed molecular studies that were previously only possible in animal systems or eggs.

Question 7

why was poliovirus such a good model virus to use

Answer 7

because it has such a robust replication cycle making it easily cultured (a lot about what we know about + sense RNA viruses comes from polio virus)

Question 8

How did culturing poliovirus in lab cells advance virology research?

Answer 8

Culturing poliovirus in lab cells enabled a new era in virology by allowing controlled experiments and advancing our understanding of RNA virus replication and biology.

Question 9

What is the current status of the poliovirus eradication campaign?

Answer 9

The poliovirus eradication campaign has made significant progress, bringing the virus close to eradication, though it has not been fully eliminated yet.

Question 10

What makes rhinoviruses important in terms of public health?

Answer 10

Rhinoviruses are a major cause of the common cold and are among the most widespread microbial infections, frequently responsible for respiratory symptoms like coughing and sneezing.

Question 11

What historical significance does the Foot-and-Mouth Disease virus have in virology?

Answer 11

The Foot-and-Mouth Disease virus holds historical significance as the first animal virus ever discovered, in 1897 marking a foundational moment in the field of virology.

Question 12

What is RNA-dependent RNA polymerase, and why is it important in RNA viruses like poliovirus?

Answer 12

an enzyme that synthesizes RNA from an RNA template. It’s essential for RNA viruses like poliovirus because eukaryotic cells do not naturally replicate RNA, so the virus must carry or encode this enzyme to replicate its genome.

Question 13

when was RNA dependent RNA polymerase discovered

Answer 13

1960s

Question 14

How does the concept of a polyprotein benefit viruses like poliovirus?

Answer 14

The polyprotein strategy allows viruses to encode their entire proteome in a single large protein, which is then cleaved by viral proteases into functional units. This approach offers genetic economy, enabling viruses to maximize protein production from limited genome space.

Question 15

What are viral proteases, and what is their role in Picornaviruses?

Answer 15

Viral proteases are enzymes encoded by the virus that cleave the polyprotein into individual functional proteins. This process is crucial for viral replication and protein functionality in Picornaviruses.

Question 16

What are infectious cDNA clones, and how were they used with Picornaviruses?

Answer 16

Infectious cDNA clones are DNA copies of viral genomes that can be manipulated in the lab and then transcribed to produce infectious virus particles. Picornaviruses were the first group of viruses for which infectious cDNA clones were developed, allowing detailed genetic studies.

Question 17

What structural biology milestone was achieved using poliovirus and rhinoviruses?

Answer 17

Poliovirus and rhinoviruses were among the first whole viruses whose structures were determined using X-ray crystallography. This was a landmark in structural virology, providing insights into viral architecture and mechanisms of infection.

Question 18

Why is viral classification considered to be in a constant state of flux?

Answer 18

Viral classification is constantly evolving due to advances in sequence-based methods, such as metagenomics, which allow scientists to discover and categorize new viruses. As a result, classification systems frequently change, and different sources may present different groupings.

Question 19

How has the classification of rhinoviruses changed over time?

Answer 19

Rhinoviruses were previously classified in a separate genus from enteroviruses but they are now grouped together due to updated sequence-based classfications`

Question 20

What are the key structural characteristics of Picornaviruses?

Answer 20

They are small, non enveloped viruses (naked) ~30nm in diameter with an icosahedral capsid composed of 60 protomers. Each protomer consists of 3 proteins : VP1, VP2 and VP3

Question 21

What type of genome do picornaviruses have

Answer 21

They have a small, positive sense, ssRNA genome ranging from ~6700 - 10,000 nt in length

Question 22

Where in the cell do Picornaviruses replicate?

Answer 22

Entirely in the cytoplasm

Question 23

Which four proteins make up the Picornavirus capsid, and where are they located?

Answer 23

The Picornavirus capsid is composed of four proteins: VP1, VP2, and VP3, which are located on the outer surface, and VP4, which lies beneath the surface and interacts with the viral RNA.

Question 24

How do VP1, VP2, and VP3 contribute to the capsid architecture of Picornaviruses?

Answer 24

VP1, VP2, and VP3 form the external face of the virus capsid and collectively assemble into protomers, which are repeated 60 times to form the full icosahedral virus particle.

Question 25

What is the “canyon” structure found in Picornaviruses, and what is its function?

Answer 25

The “canyon” is a groove-like depression on the surface of the Picornavirus capsid that serves as the binding site for host cell receptors. It plays a critical role in viral attachment and entry into host cells.

Question 26

How do Picornavirus receptors interact with the canyon structure?

Answer 26

Host cell receptors, which are typically long, type I glycoproteins, fit into the canyon structure of the virus capsid, initiating the infection process. Different Picornaviruses use different receptors, such as the poliovirus receptor for poliovirus.

Question 27

What is the potential immune evasion advantage of the canyon structure in Picornaviruses?

Answer 27

The canyon structure may protect the receptor binding site from being easily targeted by antibodies, providing some degree of immune evasion. However, some antibodies can still access these sites with their flexible arms.

Question 28

In what region of the canyon do the viral receptors interact

Answer 28

with the pocket region which contains a lipid molecule known as the pocket factor, that contributes to the structural stability of the virus. When a host receptor binds, the pocket factor is displaced, triggering conformational changes that help the virus enter the host cell.

Question 29

How do antiviral drugs interfere with the pocket factor mechanism in Picornaviruses?

Answer 29

Antiviral drugs can be designed to mimic or displace the pocket factor, binding irreversibly in the canyon pocket and preventing receptor interaction. This blocks the conformational changes necessary for virus entry, thereby inhibiting infection.

Question 30

What were "WIN compounds" and what was the significance?

Answer 30

WIN compounds were early antiviral drugs developed in the 1980s that bound to the pocket in the rhinovirus canyon, displacing the pocket factor. They were among the first examples of structure-based rational drug design, especially effective in vitro (test tube).

Question 31

What role does VP4 play in the Picornavirus structure?

Answer 31

VP4 is located beneath the viral capsid surface and interacts with the viral RNA, likely playing a role in genome packaging and possibly in viral uncoating during cell entry.

Question 32

Why did early antiviral efforts with WIN compounds against rhinoviruses decline?

Answer 32

worked well in the test tube, but had limited clinical success especially when they were given topically ( e.g. nasal sprays/ creams) as they had many side effects.

Question 33

Why is there renewed interest in antiviral drug development for Picornaviruses now?

Answer 33

It is now recognised that rhinoviruses can cause more than just the common cold (e.g., exacerbation of asthma or COPD). and there is a push for polio eradication highlighting the need for antiviral back-up treatments in case vaccine strategies fall short or outbreaks occur.

Question 34

How does the concept of “rational drug design” apply to Picornaviruses?

Answer 34

Rational drug design involves using detailed structural knowledge of the virus to create drugs that precisely target functional sites, such as the canyon pocket. This approach allows for targeted inhibition of viral entry or replication.

Question 35

how small does the picornavirus measure

Answer 35

~ 27-30nm

Question 36

what is a picornavirus protomer

Answer 36

A unit that makes up the viral capsid - each protomer comprises of 3-4 proteins (VP1,2,3,4)

Question 37

What impacts the general shape of the virus and affects how it interacts with receptors and antibodies?

Answer 37

The interaction of the protomer proteins on the virus surface

Question 38

What structural features are common across Picornaviruses like poliovirus, EV-A71, EV-D68, RV-C15a, and hepatitis A virus? (EV = Enterovirus, RV = Rhinovirus)

Answer 38

All these Picornaviruses have a tight icosahedral capsid, are non-enveloped, and have a size of approximately 30 nm. They all contain a positive-sense single-stranded RNA genome, and most have a canyon structure for receptor binding.

Question 39

How does Rhinovirus C15a differ structurally from other Picornaviruses like hepatitis A virus?

Answer 39

Rhinovirus C15a tends to have more protruding surface features, making it appear structurally more irregular compared to the smoother capsid of Hepatitis A virus. Additionally, some RV-C types lack the typical canyon structure.

Question 40

What is the significance of the canyon structure in Picornaviruses, and do all of them have it?

Answer 40

The canyon structure facilitates receptor binding, critical for cell entry. While many Picornaviruses (like poliovirus, EV-A71, EV-D68) possess it, some Rhinoviruses (e.g., RV-C15a) may lack a defined canyon, showing that receptor interaction mechanisms can vary.

Question 41

Describe the genome structure of Picornaviruses.

Answer 41

Picornaviruses have a positive-sense ssRNA genome ranging from 6.7 to 10.1 kb. The genome contains a single large open reading frame (ORF) that encodes a polyprotein. It is polyadenylated at the 3′ end similar to eukaryotes and has a viral protein (VPg) linked to the 5′

Question 42

Why is the 3′ poly-A tail important for Picornaviruses?

Answer 42

The poly-A tail at the 3′ end mimics eukaryotic mRNA, enhancing stability and translation efficiency by facilitating interaction with host translation machinery.

Question 43

What is the function of the VPg protein at the 5′ end of the Picornavirus genome?

Answer 43

VPg (Viral Protein genome-linked) is a small viral protein that serves multiple roles: Acts as a primer for RNA synthesis during replication. Replaces the typical 5′ cap in translation initiation. Plays a role in packaging and genome stability.

Question 44

How many aa long is the single polyprotein encoded

Answer 44

2100 - 2400 aa long

Question 45

How does the polyprotein strategy benefit Picornaviruses?

Answer 45

It allows genetic economy—maximizing output from a compact genome. Producing one long polyprotein avoids complex transcriptional regulation and enables coordinated synthesis of all necessary viral proteins.

Question 46

Why is the single ORF strategy unusual compared to eukaryotic gene expression?

Answer 46

In eukaryotic cells, one mRNA typically encodes one protein (monocistronic). In contrast, Picornaviruses use a polycistronic-like strategy by processing one polyprotein into multiple proteins, which is rare in eukaryotic systems.

Question 47

What does the 5' UTR act as

Answer 47

an Internal Ribosome Entry Site (IRES) which recruits translational machinery so the ribosome can initiate translation / protein synthesis without a 5' cap (cap-independent translation)

Question 48

What is the hallmark feature of positive-sense RNA viruses like Picornaviruses?

Answer 48

Their RNA genome is infectious on its own. When introduced into a cell, it acts as mRNA, initiating translation without requiring additional enzymes.

Question 49

How does this differ from negative-sense RNA viruses like influenza?

Answer 49

Negative-sense RNA viruses cannot be directly translated. They must first be transcribed into positive-sense RNA by viral RNA-dependent RNA polymerase, which is packaged in the virus particle.

Question 50

What happens to the single polyprotein after it is synthesised

Answer 50

It is cleaved by proteases into structural (P1 - Capsid) and non structural (P2 and P3 - Proteases and RNA synthesis) proteins

Question 51

What is the general layout of the Picornavirus genome?

Answer 51

1. 5′ UTR (with IRES) 2. Structural proteins (VP1–VP4) near the 5′ end 3. Non-structural proteins (2A, 2B, 2C, 3A, 3B (VPg), 3C^pro, 3D^pol) toward the 3′ end 4. 3′ UTR with a poly-A tail

Question 52

What do the structural and non-structural proteins do?

Answer 52

Structural proteins (VP1–VP4) form the viral capsid. Non-structural proteins are involved in polyprotein cleavage (2A, 3C^pro), RNA replication (3D^pol), membrane rearrangement, and RNA binding.

Question 53

Do other viruses besides Picornaviruses use polyproteins?

Answer 53

Yes. Other positive-sense RNA viruses like Hepatitis C virus (HCV), dengue virus, and even coronaviruses use polyprotein strategies, though coronaviruses are more complex. HIV (a retrovirus) also synthesizes polyproteins that are cleaved by viral protease.

Question 54

How do picornaviruses enter host cells?

Answer 54

They bind to specific host receptors (with their groove type I glycoproteins), but the exact mechanism of entry and uncoating remains unclear due to its transient nature. Entry may occur via: Endocytosis (entire virus enters via endosome), or direct genome release (capsid opens a channel at the membrane).

Question 55

Why is viral entry difficult to study?

Answer 55

Because it happens very quickly and involves small, transient intermediates that are hard to capture experimentally.

Question 56

What happens once the viral genome enters the cytoplasm?

Answer 56

The positive-sense RNA acts like host mRNA and is immediately translated into one large polyprotein.

Question 57

What happens when the virus enters into the cytoplasm

Answer 57

You get : 2. Uncoating of the viral RNA genome into the host cytoplasm where it acts as mRNA 3. Translated as host mRNA into polyproteins 4. proteolytic processing whereby the polyprotein is cleaved to produce individual viral proteins : structural proteins or non structural proteins 5. RNA synthesis occurs on membrane vesicles induced by viral proteins where the viral + strand RNA is copied by the viral RNA polymerase to form full length - strand RNAs 6. - strand RNA is copied to produce additional + strand RNA (positive strand amplification) 7. newly synthesised + strand RNA is translated to produce additional viral proteins 8. Structural proteins are used for genome packaging 9. Release of packaged genome and cell lysis

Question 58

How is the RNA genome replicated?

Answer 58

Positive-strand RNA → transcribed into a negative-sense RNA Negative-strand RNA → serves as a template for making new positive-sense RNAs

Question 59

What causes formation of membrane vesicles in infected cells?

Answer 59

Viral proteins hijack host lipid synthesis and membrane structures to create replication organelles.

Question 60

How are picornaviruses typically released from host cells?

Answer 60

They are cytolytic—they lyse (burst open) the host cell, releasing mature virus particles.

Question 61

What is the impact of cytolysis in infections like poliovirus?

Answer 61

It causes host cell death, contributing to tissue damage and disease symptoms (e.g., in poliovirus, destruction of motor neurons).

Question 62

Do all polioviruses replicate at the same rate or in the same way?

Answer 62

No. Some are highly cytolytic and rapid, while others are more stealthy, replicate slowly or persist in cells for longer ... this highlights that while there are shared core mechanisms, each virus has unique traits that affect pathogenicity, persistence, and immune evasion.

Question 63

What are the two main viral proteases involved in polyprotein cleavage?

Answer 63

2A proteases and 3C protease ( or 3CD protease when in precursor form)

Question 64

What type of cleavage does 2A protease perform?

Answer 64

Co-translational cleavage – as the ribosome synthesizes 2A, it cleaves itself from the polyprotein (specifically from P1).

Question 65

What is cis cleavage in the context of viral proteases?

Answer 65

The protease cleaves within the same molecule (e.g., 3C protease cleaving sites near itself on the polyprotein). "3C autocatalytic "cis" cleavage"

Question 66

What is trans cleavage?

Answer 66

The protease cleaves separate molecules or regions (e.g., 3C / 3CD protease cleaving other proteins elsewhere in the polyprotein).

Question 67

What determines the cleavage sites?

Answer 67

Specific short amino acid sequences (usually 3-6 residues long), not random - each protease recognises defined motifs

Question 68

Why is 3CD protease important

Answer 68

It’s a precursor protein with both 3C protease and 3D RNA-dependent RNA polymerase polymerase domains and can cleave in ways 3C alone cannot, providing more regulatory flexibility.

Question 69

What is the advantage of using a polyprotein strategy?

Answer 69

- It allows viruses to encode multiple proteins from a single RNA. - Provides temporal control over protein production. - Enables efficient genome usage in a small RNA genome.

Question 70

how does co translational cleavage and cleavage of the polyprotein benefit viruses

Answer 70

- cleavage of the polyprotein regulates timings of protein activity which is important for different stages of the virus lifecycle which can influence pathogenicity and the establishment of infection within a host. - helps condense the genome allowing for more efficient packaging within the viral particle - the cleavage of the polyprotein is controlled by proteases that regulate viral replication - the intermediates have varying half lives within the cell (affecting their concentration levels)

Question 71

What do viral cleavage intermediates do?

Answer 71

They act as functional proteins for short windows of time before being further cleaved into mature proteins.

Question 72

How is the timing of protein production controlled?

Answer 72

Different cleavage site sequences control cleavage speeds e.g. 3C vs. 3CD have different specificities, giving control over when intermediates are processed.

Question 73

why is delayed production of structural proteins beneficial

Answer 73

Prevents premature genome encapsidation, which would stop further replication. Ensures mass replication before virion assembly.

Question 74

What does 2C protein do?

Answer 74

Likely acts as a helicase, helping unwind RNA during replication or packaging (also contributes to membrane remodeling)

Question 75

What is the role of 2A protease?

Answer 75

Starts cleavage co-translationally (cuts off P1). Disrupts host cell translation by cleaving eIF4G.

Question 76

What did scientists notice about rates of protein synthesis after polioviral infection

Answer 76

Infected cells experienced a significant decrease in protein synthesis rates after 2.5 hours of infection (nearly halting replication within the cell). After 5 hours almost all the proteins being synthesised are viral proteins. These findings were based on quantifying protein levels using polyacrylamide gel

Question 77

how can picornaviruses shut down host protein synthesis but still maintain their own

Answer 77

The virus's 2A protease recognises and cleaves the host protein eIF4G. This shits down CAP-dependent translation, reducing host protein synthesis as the mRNA can no longer be translated. This virus bypasses this by using the 5' IRES which allows ribosomes to directly bind to the viral RNA and translate viral proteins

Question 78

what does elF4G typically do?

Answer 78

typically recruits eIF4E and the 40S ribosomal subunit to the mRNA, interacting with other initiation factors to assemble the pre-initiation complex.

Question 79

ways picornaviruses can avoid detection

Answer 79

1. Capable of surviving the highly acidic, non-surveyed conditions of the stomach, so they can spread through the lymphatic system 2. fast replication: the virus can overwhelm host organs before any immune response is mounted 3. The canyons for attachment on the viral surface are too narrow for antibody access, so are protected

Question 80

How are enteroviruses inactivated

Answer 80

by chlorine, UV and temperatures above 50*c

Question 81

how can Picornaviruses avoid apoptosis

Answer 81

Viral proteases e.g. 2A and 3C, can cleave pro-apoptotic factors e.g. Caspase 9 so it cant get activated or initiate the apoptotic cascade - this ensures optimal conditions for their replication and spread

Question 82

How are Enteroviruses transmitted?

Answer 82

fecal-oral route

Question 83

how long are Enteroviruses infectious for

Answer 83

for several days at room temperature and are even stable at room temperature and particularly stable at pH 2 (except rhinoviruses)

Question 84

What are some of Enteroviruses (A-J) species examples

Answer 84

1. Inflammatory heart disease 2. Pancreatitis 3. Hand, foot and mouth disease 4. Acute haemorrhagic conjuctivitis 5. type I diabetes ( strongly linked) 6. Febrile illness (most common) - fever 7. menengitis ... many more

Question 85

how many Enterovirus infections are there in the USA each year

Answer 85

30-50 million

Question 86

what is the asymtomatic infection rate for Enterovirus (A-J)

Answer 86

over 90% (this is very high)

Question 87

What are some major clinical features of enteroviruses (A-L ... in the 10% who do get symtoms

Answer 87

- They enter through the oropharyngeal or intestinal mucosa - secretory IgA can prevent infection - They spread by viraemia (blood) to target tissues such as the skin, muscle, brain and meninges ( layer that protects the brain and spinal chord) - The virus is shed in faeces

Question 88

Which virus infects the skin and what does this present as

Answer 88

You get hand-foot-and-mouth disease which can lead to rash formation

Question 89

Which enterovirus infects the muscle and what does this present as

Answer 89

Echovirus or coxsackie A and B viruses. If this is in the heart this can lead to Myocarditis or in the thorax it can lead to pleuridynia

Question 90

Which virus infects the brain and what does this present as

Answer 90

Poliovirus or coxsackie A and B viruses. This an lead to paralysis and encephalitis

Question 91

Which virus infects the meninges (in the brain) and what does this present as

Answer 91

Echovirus, poliovirus, coxsackie A and B viruses. This can lead to meningitis

Question 92

Which Enterovirus is suggested to be linked with the induction or exacerbation of T1D

Answer 92

coxsackievirus infection

Question 93

How does epidemiological data suggest coxsackievirus infection induces or exacerbates T1D

Answer 93

1. The virus infects pancreatic B cells where it causes stress marker up-regulation 2. APC engulf infected B cells 3. APC secrete inflammatory cytokines and present beta cell antigens 4. if enough autoreactive T cells are present, autoimmune beta cell destruction occurs, leading to T1D.

Question 94

What were the findings regarding the IFH1 gene in the study of 480 healthy and T1D patients?

Answer 94

4 variants in the IFH1 gene independently lowered the risk of developing T1D by 50%.

Question 95

What is the role of the IFH1 gene in relation to picornavirus infection and T1D?

Answer 95

The IFH1 gene is involved in sensing picornavirus infection and inducing an interferon response. This leads to upregulation of MHC I, which enhances the presentation of antigens, allowing T cells to recognize and destroy infected cells. Mutations in the IFH1 gene impair this response, making cells less susceptible to T cell destruction.

Question 96

What happens to most individuals following an infection with Enterovirus (beta cells)

Answer 96

anti-viral defences were induces to prevent the development of a sustained and productive infection of the beta cells and the virus gets cleared

Question 97

What happens some individuals (mostly neonates or those with impaired anti-viral defences) following an infection with HEV in beta cells

Answer 97

In these individuals, the enterovirus infects the beta cells and establishes a productive lytic infection. This can lead to the release of free virus or viral and beta cell-specific antigens, which, in genetically predisposed individuals, may trigger the activation of autoreactive immune cells, potentially resulting in Type 1 Diabetes.

Question 98

What happens in some individuals when their host anti-viral defense only partially inhibits viral replication following an infection with HEV in beta cells?

Answer 98

A persistent infection may develop, leading to upregulation of HLA-I (human leukocyte antigen class I) and enhanced presentation of both beta cell and viral antigens on the cell surface. In at-risk individuals, this can trigger autoimmune responses by autoreactive immune cells. The virus may also spread to other cells via extracellular vesicles (still under investigation for human beta cells).

Question 99

What is associated with a persistent HEV infection?

Answer 99

A persistent HEV infection is associated with a 5' UTR (untranslated region) deletion of the viral genome and the formation of double-stranded RNA (dsRNA).

Question 100

What can dsRNA activate in the host?

Answer 100

Double-stranded RNA (dsRNA) can activate host pathogen recognition receptors, stimulating an enhanced interferon signature. This leads to the upregulation of HLA-I and enhances the presentation of beta cell and viral antigens on the cell surface.