Richard Kingston Topic 2

Question 1

What is the difference between a receptor and an attachment factor for viral infection?

Answer 1

A receptor is a molecule which mediates adherence of viral particles to the cell surface, leading to subsequent internalization of the virus while an attachment factor will not lead to virus internalization

Question 2

What is the difference between a susceptible cell and a permissive cell in virology?

Answer 2

A susceptible cell carries the receptor for viral entry while a permissive cell allows replication once the virus gains entry.
A cell may be either of these things or both

Question 3

How can receptor binding influence the infectivity of a virus?

Answer 3

As the virus will require its receptor to be present it will determine the host range and also cause tissue tropism in the virus

Question 4

How does poliovirus illustrate how receptors can influence the host range of a virus?

Answer 4

Poliovirus uses an immunoglobulin-like molecule CD155 as its receptor this molecule is only found in primates and old world monkeys so poliovirus can only infect these groups. This was illustrated as experiments done with recombinant mice expressing CD155 can be infected by poliovirus showing that mouse cells are permissive and just naturally lack the receptor

Question 5

How does the HIV virus demonstrate tissue tropism?

Answer 5

HIV uses CD4 as its receptor this is found on the surface of lymphocytes particularly helper T cells as well as macrophages. These cells are key to the adaptive immune system and elimination of them by HIV allows secondary pathogens to flourish

Question 6

What virus illustrates a distinct lack of tissue tropism and host range due to its receptor?

Answer 6

Nipah and Hendra viruses which are zoonotic paramyxoviruses. Their natural reservoir is bats however they are readily transmitted to other species where they cause fatal respiratory disease and/or encephalitis. They spread throughout the body including multiple organ systems this is due to their use of ephrin B2/B3 as receptors which are highly conserved between species and have widespread cellular distribution particularly in vascular endothelial cells and neurons

Question 7

What are co-receptors?

Answer 7

Some viruses require binding to more than one type of molecule on the cell surface to mediate their entry to the cell.
HIV is an example of this as CD4 binding is not sufficient for cell entry. With the chemokine receptors CXCR4 or CCR5 being obligate co-receptors.

Question 8

What is the difference between a receptor and a co-receptor?

Answer 8

There is no true difference; it is mainly an arbitrary distinction with the first molecule to be found termed the receptor.

Question 9

How does measles illustrate how a virus can use multiple different receptors?

Answer 9

Measles principally uses signalling lymphocyte activation molecule (SLAM) as its receptor expressed exclusively on immune cells. This allows spread of the virus via alveolar macrophages and dendritic cells and causes immunosuppressive activity.
However it uses Nectin-4 (an immunoglobulin like molecule in adherens junctions) to initially infect respiratory epithelium

Question 10

What is the receptor used only by the attenuated measles strain used in the vaccine?

Answer 10

The mutated H protein of the vaccine strain allows the virus to bind to CD46 which is a ubiquitously distributed cellular protein. This may explain why this strain is attenuated as CD46 can act as a decoy receptor misleading the vaccine strain but not wild type measles

Question 11

What is sialic acid?

Answer 11

Many cell surface proteins and lipids carry covalently linked oligosaccharides. Sialic acid is often found on the cap of the sugar chains for glycoproteins and glycolipids. As a result it is found at the extremities of many molecules found on the cell surface.

Question 12

How does sialic acid act as a receptor for viruses?

Answer 12

The sialic acids used by viruses as receptors are typically attached to a penultimate glucose via an alpha 2,3 or alpha 2,6 linkage. The most well-known virus which uses this molecule as a receptor is influenza which uses hemagglutinin to bind to sialic acid and initiate entry and neuraminidase to cleave the sialic acid allowing release of newly generated viral particles

Question 13

How can sialic acid cause host range restriction for the influenza virus?

Answer 13

Humans preferentially use sialic acids with an alpha2,6 linkage causing a bent conformation
Avians preferentially use sialic acids with an alpha 2,3 linkage with a more linear structure.
This typically prevents avian flu strains from infecting humans unless there is a mutation

Question 14

What are the differences between viral entry for enveloped and non-enveloped viruses?

Answer 14

For non-enveloped viruses entry typically involves pore formation or membrane rupture while for enveloped viruses it typically involves membrane fusion
These events may take place at the cell surface or they may occur later on such as in the endosome etc

Question 15

How does endocytosis play a role in viral entry?

Answer 15

Many enveloped and non-enveloped viruses get transported deep inside the cell via endocytosis, typically receptor mediated endocytosis, with the most common uptake pathway being the clathrin mediated pathaway

Question 16

How do the pH changes in the endosomal network play a role in viral entry?

Answer 16

Once internalised within a primary endocytic vesicle the virus will enter the endosomal network consisting of early endosomes, maturing endosomes, late endosomes and recycling endosomes. pH decreases as the virus proceeds through this system which may lead to conformational changes in viral proteins

Question 17

How does membrane fusion for viral entry occur?

Answer 17

This is a non-spontaneous process involving fusion proteins carried by the virus. These proteins have fusion peptide which they extend into the host membrane in an extended intermediate, this intermediate then collapses due to the back folding of the C-terminal segment of the protein resulting in the membrane. Eventually the intermediate will collapse far enough to bring the membranes into contact with each other forming a hemifusion stalk. The hemifused bilayers then open up into a fusion pore and the C-terminal ectodomain snaps the protein into its post fusion position preventing pore resealing

Question 18

How does membrane fusion offer a promising new target for anti-viral drugs?

Answer 18

Virus-cell membrane fusion requires a change for positive to negative membrane curvature, this can be targeted by rigid amphipathic fusion inhibitors which insert onto the outer leaflet of the viral membrane, this results in the energy required to form the stalk necessary to fuse with cell membranes being too high for the virus stopping the viral replication cycle

Question 19

What are the three mechanisms used by non-enveloped viruses to breach membranes?

Answer 19

Transient modification of the cellular membrane, pore formation and total disruption of the limiting membrane

Question 20

What is the mechanism used by parvoviruses to breach membranes?

Answer 20

Due to the low pH of the endosome parvoviruses use a phospholipase A2 domain which allows membrane passage via a temporary modification of the endosomal bilayer

Question 21

What is the mechanism used by reovirus to breach membranes?

Answer 21

Triggered by the endocytic protease cathepsin B, reovirus releases the Micro-1N peptide which forms size selective pores in the endosomal membrane. It is unclear as to if this change is linked to osmotic lysis of the vesicle

Question 22

What is the mechanism used by adenovirus to breach membranes?

Answer 22

Triggered by the low pH of the endosome adenovirus protein VI inserts itself into the luminal leaflet of the endosomal membrane inducing positive curvature and total fragmentation of the endosome

Question 23

Can passive diffusion account for viral transport within a cell?

Answer 23

No as passive diffusion is too slow, particularly in the crowded environment of a cell

Question 24

How can the cytoskeleton cause viral transport within a cell?

Answer 24

Some viral particles or sub-viral assembly binds directly to the cytoskeleton transport machinery this commonly includes dynein which moves towards the minus end of the microtubule which is towards the cell centre or kinesin walks in the opposite direction

Question 25

How do adenoviruses use the cytoskeleton for transport?

Answer 25

After exiting the endosome adenoviruses are transported via microtubules to reach the nucleus with a hexon on the capsid surface binding to dynein

Question 26

How are herpesviruses and papillomaviruses transported in the cell?

Answer 26

These bind directly to dynein to achieve microtubule based transport

Question 27

What is the adenovirus entry pathway?

Answer 27

The virus binds through coxsackie and adenovirus receptor on the cell plasma membrane causing it to be taken up via endocytosis where some capsid proteins are lost. After endosomalysis, adenoviruses will then move bidirectionally along the microtubules using dynein for transport towards the nucleus where it binds to the nuclear pore complex allowing the viral genome to be injected into the nucleus for viral reproduction

Question 28

How can retrograde transport of viruses occur without direct interaction with dynein?

Answer 28

They may be transported large distances within endocytic vessels piggy-backing those methods of transport

Question 29

What is an example of the actin cytoskeleton being used to transport viruses

Answer 29

The fliovirus, Marburg virus is a close relative of Ebola and can cause hemorrhagic fever in humans. This virus can be transported within the cell at velocities of up to 400 nm/s

Question 30

What is the one family of DNA containing viruses which does not replicate in the nucleus?

Answer 30

The pox family

Question 31

How the majority of DNA containing viruses entry to the nucleus for their replication?

Answer 31

Via nuclear pore complexes

Question 32

How does murine leukemia virus gain entry to the nucleus?

Answer 32

When the cell goes through mitosis when the nuclear envelope is temporarily disassembled

Question 33

How do HIV-1 and influenza A gain entry to the nucleus?

Answer 33

Both these viruses undergo extensive disassembly in the cytoplasm, the released subparticles contain nuclear localization signals and can therefore hijack host machinery to be transported to the nucleus

Question 34

How can the nuclear pore complex allow entry of viruses into the nucleus?

Answer 34

Some viruses attach to the cytoplasmic side of the nuclear pore complex. Docking triggers particle disassembly and genome ejection allowing transport into the nucleus
Some viruses like hepatitis B virus are small enough to cross the pore complex intact

Question 35

How do parvoviruses enter the nucleus?

Answer 35

They do not use the nuclear pore complex to deliver their genome into the nucleus and instead transiently disrupt the nuclear envelope and lamina entering the nucleus through the gaps created

Question 36

Where does uncoating typically occur for enveloped viruses?

Answer 36

When viral and cellular membranes fuse which may occur at the plasma membrane or within intracellular vesicles

Question 37

Where does uncoating occur for naked icosahedral viruses?

Answer 37

It is largely unclear but it may be a process which is primed by receptor binding and acidification in the endosome

Question 38

What is an example of a virus that never fully uncoats?

Answer 38

Non-enveloped reo-viruses have a dsRNA genome and mRNA synthesis occurs within a subviral particle known as the core with newly synthesized nucleic acids extruded through channels in the spikes which decorate the core surface

Question 39

What are the most common type of viruses that replicate in the cytoplasm?

Answer 39

Positive sense single stranded RNA viruses

These viruses also typically compartmentalize their genome replication and transcription in organelle like structures

Question 40

What are replication factories?

Answer 40

Novel infection introduced vesicles produced by viruses which enhance replication efficiency by only releasing positive sense genomes (and retaining the negative sense intermediates) and protect the virus from host defenses
They are commonly formed from invaginations of ER or organellar membranes

Question 41

How do positive sense RNA viruses replicate their genome?

Answer 41

The viral genome can act as an mRNA upon infection allowing the production of viral proteins including replication proteins, this replication requires a negative sense RNA intermediate

Question 42

What is an example of a virus which forms replication factories?

Answer 42

The flockhouse virus which has a genome containing only 4 genes with only one (protein A) being required for genomic RNA replication, this is an RNA-dependent RNA polymerase
This protein locates itself to the interior of the vesicles where it recruits the viral genome, the negative sense intermediate strands are retained within the replication factor while the progeny positive sense RNAs are exported

Question 43

What are the necessary steps a progeny virion must go through in order to exit the host cell?

Answer 43

Following synthesis, viral proteins and nucleic acids may need to be transported to the site of assembly
The viral structural proteins need to assemble into a capsid or nucleocapsid, packaging the viral genome and incorporating and associated replication machinery
Enveloped viruses formation of internal structures of the virion must be co-ordinated with acquisition of a cellular membrane which has had viral proteins inserted

Question 44

Do all non-enveloped viruses escape the cell via lysis?

Answer 44

No while this is largely true, the area is not well understood but there is clear evidence to suggest Bluetongue virus and poliovirus can escape through non-lytic methods

Question 45

How do enveloped viruses leave the cell?

Answer 45

The complete their replication cycle via budding, this process may occur after or simultaneously with virion particle formation

Question 46

What is membrane fission?

Answer 46

In order for enveloped viruses to leave the cell they must deform the membrane which is driven by either envelope proteins or internal viral components. The membrane must then be pinched off in a membrane fission event. This event is not spontaneous and uses the ESCRT pathway

Question 47

What is the ESCRT pathway?

Answer 47

A pathway that exist in all eukaryotes and is typically used to create vesicles which bud into late endosomal compartments known multivesicular bodies. This pathway can be used viruses like retroviruses to achieve membrane fission

Question 48

How did the retroviral particle lend insight into how membrane fission occurs?

Answer 48

The major structural proteins of a virus are produced as the GAG polyprotien. These assemble into a spherical capsule pushing through the cellular membrane. This however does not complete the membrane release. In order to complete this process the Late domains which are short sequence motifs within the GAG polyprotein were required. These late domains were later discovered to recruit cellular proteins

Question 49

What are the Late domains identified through studies of retroviral particles?

Answer 49

PTAP domains which recruit cellular TSG101
YP(X)nL domains which recruit ALIX
PPxY which recruit Nedd4
These cellular proteins are all part of the ESCRT pathway

Question 50

How do viruses use the ESCRT pathway for membrane fission?

Answer 50

In the HIV model the L-domains in the C terminus of Gag engages the ESCRT machinery leading to the assembly of an ESCRT-III tube with a dome-like membrane binding surface within the neck of the budding virion. This causes constriction of the bud neck until spontaneous fission occurs

Question 51

What is an example of a virus which does not use the ESCRT pathway to escape the host cell?

Answer 51

Influenza which uses budding and membrane scission depending on the virally encoded ion channel (the M2 protein)

Question 52

Does the virus replication cycle require an extracellular phase?

Answer 52

No there are instances when a virus can spread directly from one cell to another such as seen in the HIV virological synapses

Question 53

How does cell to cell spreading of HIV-1 occur?

Answer 53

If an uninfected immune cell transiently adheres to an infected cell a special structure known as the virological synapse forms between the cells. The synapse contains HIV receptors which have localised to a filopodia which has been corrupted by the virus. These filopodia help to capture cells and then mature into the stable contacts of the virological synapse