Lecture 4 and 5

Question 1

What did Enders, Weller and Robbins do in 1942?

Answer 1

They discovered cell culturing by culturing primary embryonic (non-neuronal) cells

Question 2

How did Enders, Weller, and Robbins culture the cells?

Answer 2

They were cultured on plastic surface (most commonly) or as liquid suspensions (bioreactors)
Large quantities, enabling studies of structure, chemical composition, development of vaccines

Question 3

What are cell cultures used for?

Answer 3

Widely used in research and vaccine development

Question 4

How did cell cultures play a major role in virology research?

Answer 4

Vaccines against polio, measles, and rubella were derived from cell culturing
Discovery of oncoviruses
Discovery of reverse transcriptase (Baltimore & Temin) –> (Discovered at same time, but independently)
Plaque assay and the “One step growth experiment”
Virus replication cycle

Question 5

What are the three types of cell cultures? Be sure to explain them

Answer 5

Primary cell cultures: Derived from live tissues/organs, composed of multiple cell types, limited capacity in cell division: 5- 20 times –> Not homogeneous
–> Monkey kidney: used for developing polio vaccine, chicken embryo, mouse embryo
‘Diploid’ cell strains: Single cell type: mainly epithelial or fibroblast; cell division: up to 100 times. Normal morphology, number of chromosomes
Continuous (aka immortal cell lines: Homogenous in cell type, immortal, infinite capacity of cell division, abnormal chromosome morphology & number, loss of contact inhibition: detach from surface, piling up, forming ‘focus’ –> Tumorigenic: produce tumours when implanted in lab animals. Source: tumours, transformed cell strains, cells mutated by oncoviruses or mutagens. Most commonly used, never die –> continuously divide and often cancer cells
HELA

Question 6

What are Cytopathic effects, or CPE?

Answer 6

CPE: morphological alterations of a cell due to viral infection
Cell death, often from cytotoxic viruses
Rounding up & detaching from surface
Syncytium: large, multi-nucleate cell bodies due to fusion
–> Form large cells, induces fusion of many cells, many cells come together or rather, fuse together
Alterations in the morphology and the number of chromosomes
Inclusion Bodies:
—> Polyhedron inclusion bodies (PIBs)
—> ‘Negri’ bodies (rabies virus-infected cells)
—> Inclusion bodies for many plant viruses (X-bodies, pinwheels, cytoplasmic inclusion bodies, multi-vesicular bodies)

Question 7

What are the differences between an animal and plant cells?

Answer 7

Plant cells have a cell wall and chloroplast, and they are very different cells:
The way viruses enter each cell is completely different

Question 8

Explain the One-step growth cycle

Answer 8

Critical: synchronous infection of cells with viruses; require high MOI: 5-10
Monitor virus growth at a set of intervals (time course experiment)
Add In growth curve

Question 9

Explain the parts of the one step growth cycle

Answer 9

1) Eclipse Period: from absorption to appearance of first intra-cellular virion
2) Latent Period: time between absorption and the first extra-cellular virion
3) Burst size: the sum of virions produced in a single cell

Question 10

What is the MOI?

Answer 10

Multiplicity of Infection, which is the number of infectious virions added per cell
Not all cells in the cell culture receive the same number of viruses.
MOI of 5-10 are commonly used to achieve synchronous infection
Distribution of virions among cells is calculated by Poission distribution
An example: MOI: 5-10 means for every cell infected, there are 5-10 infectious particles

Question 11

What is attachment in the virus replication cycle?

Answer 11

Attachment is collision between viruses & cells via Brownian motion
Weak contact via interaction between viral particle and the negative charges on cell surface
The specific attachment is achieved via interactions
The attachment is strengthened when more interactions occur between attachment proteins and receptors

Question 12

What are the interactions that occur in the attachment phase?

Answer 12

1) Attachment protein on the virus –> ex. HA in influenza virus and
2) Receptor or receptors on the surface of the host cell –> ex. sialic acid for HA

Question 13

Explain the plasma membrane at the surface of eukaryotic cells

Answer 13

There are up to 500,000 potential receptor molecules per cell
Plasma membrane contains ‘lipid rafts’ with distinctive functions
Viruses hijack surface molecules and lipid rafts for entry and replication

Question 14

Where are membrane proteins are attached?

Answer 14

Some membrane proteins are attached directly to the membrane via TMD
Some membrane proteins are anchored indirectly via fatty acids or alcohols to plasma membrane
–> Myristic acid: 14 carbon fatty acid
–> Farnesyl: 15 carbon alcohol
–> GPI (glycosyl phosphatidyl inositol): linked proteins (occurs only on the external surface of the plasma membrane

Question 15

What are some examples of attachment protein & receptors for a naked virus?

Answer 15

1) Attachment via surface features of a virion:
- -> Poliovirus & rhinoviruses have a canyon (depression) surrounding each pentamer, serving as the attachment site for cell receptors
2) Attachment via fibres on virion surface:
- -> An example of this is adenoviruses
- -> Fibers: homo-trimer of finer protein, anchored at each penton base
- -> Fibres are the attachment protein here on the virion for the virus to attach
- -> Terminal knob on finer with depression: site for attaching ‘Car’

Question 16

What are some cell receptors for naked viruses?

Answer 16

1) Ig-like for RNA
2) SCR-like
3) Integrin: for adenoviruses
Remember: cell receptors are on the cell and required for virus to attach to the cell

Question 17

What are some examples of attachment protein & receptors for an enveloped virus?

Answer 17

Glycoproteins are responsible for attachment, for example: Hemagglutinin is a glycoprotein (homo-trimer) of influenza A & B
–> H spike: Responsible for attachment
–> N spike: Neuraminidase
H spike binds to sialic acid receptor in the cell, and the N spike cleaves the glycosidic bond
Viruses always have what they need
Another example is HIV-1, which is a glycoprotein on the virus:
HIV-1 binds CD4 of Th cells & macrophages
–> A co-receptor is also required for viral entry

Question 18

Explain receptors

Answer 18

Receptors for a small subset of high impact viruses have been identified, but receptors for the majority of viruses remain unknown
Many viruses share the same receptors: Ig like, sialic acid, heparin sulphate
Viruses with carbohydrate receptors tend to have broader host range
Presence of receptor & co-receptor determines, in part, the host range and tissue tropism of a given virus –> the receptor determines if the virus can enter the cell and do its job
Identified receptors represent only a small subset of surface molecules

Question 19

Entry and Uncoating: What are three basic tricks viruses use to enter a host cell? Be sure to explain

Answer 19

1) Drilling a hole at plasma membrane: Picornaviruses
2) Fusion of viral envelope with plasma membrane: Occurs at neutral pH but requires fusion proteins/peptides: paramyxoviruses, retroviruses, coronaviruses
3) Receptor-mediated endocytosis, followed by un-coating at a intracellular membrane (endosomal or nuclear): requires low pH environment: endoscope, lysosome

Question 20

What is the model for poliovirus entry?

Answer 20

There are two possible ways that polio genome RNA enters the cell:
Plasma Membrane
Endosomal Membrane

Question 21

How does the model for poliovirus entry work?

Answer 21

1) Attachment to receptor Pvr
2) N-terminus of VP1 exposed and inserted into plasma membrane
3) Pore formed and viral RNA inserted
Put in image of this

Question 22

Explain the entry & un-coating at the plasma membrane

Answer 22

Take HIV-1:
Viral entry requires binding to both receptor (CD4) and a co-receptor (CCr5 or CxCr4)
Binding to receptors triggers exposure of fusion peptide and fusion between viral and plasma membrane

Question 23

What is the cellular function of receptor-mediated endocytosis ?

Answer 23

Cellular function: selective important of extracellular molecules (ligand) into a cell through receptor & membrane invagination. Very good at highjacking cellular function

Question 24

How does receptor mediated endocytosis work?

Answer 24

Formation of clathrin coated pits requires ATP hydrolysis, and there is a gradual decrease in pH from early endoscope to late endosome, to lysosome

Question 25

How does receptor mediated endocytosis & uncoating @ endosomal membrane work for influenza?

Answer 25

1) HA attaches sialic acid
2) Endocytosis: receptor mediated
3) Fusion peptide emerges from HA, inducing membrane fusion
4) H+ is pumped into virion
5) Matrix layer dismantled
6) RNPs are released into cytosol
7) RNPs import into nucleus

Question 26

How does receptor mediated endocytosis & uncoating @ endosomal membrane work for adenoviruses (naked viruses)?

Answer 26

1) Fiber binds to Car (Receptor on cell)
2) Penton base interactions with integrin, leading to endocytosis
3) Losing fibres in endosome
4) Penton base dismantles in the late endosome
5) Broken virion release into cytosol
6) Take a free ride on microtubules
7) Dock at nuclear pore
8) Tug of war between histone & Dunedin releases viral DNA
9) Transcription & replication

Question 27

How do phages with dsDNA genomes: T4 enter the cell?

Answer 27

1) Tail fibres recognize and attach to receptors (LPS and OmpC) on bacterial cell surface, and their is an induction of conformational changes in base plate
2) There is a contraction of tail sheath to expose tube needle
3) Tube needle drills through the outermsmbrane, and the phage lysosomes dissolve the peptidoglycan layer in the periplasm
4) DNA is injected through the plasma membrane into the. cell

Question 28

What are the routes of entry of plant viruses into plant and plant cells?

Answer 28

Most plant viruses do not require receptors to enter the plant cell.
Ways plants can enter cell:
1) Mechanical transmission through minor wounds and abrasions
2) Vertical transmission via reproductive organs (pollen or seeds)
3) Vegetative propagation materials (for perennial and fruit crops)
4) Through grafting between rootstock and scion
5) Transmitted by insect vectors

Question 29

What are some important terms for the biosynthesis of the viral replication cycle?

Answer 29

Biosynthesis: synthesis of all viral components (proteins, enzymes, RNA, DNA) required for building the next generation of viruses
Transcription: production of mRNAs from genome (DNA or RNA)
Reverse transcription: generation of cDNA using RNA as template. Exists in only retroviruses and retro-like viruses, opposite of transcription: RNA –> DNA
Translation: production of polypeptides using mRNA; relies entirely on cell translation machinery
Genome replication: production of nascent viral genomes (DNA or RNA, depending on the virus)

Question 30

What is the revised Baltimore system

Answer 30

Put in picture

Question 31

What are the three broad categories of viruses based on genomes?

Answer 31

1) DNA Viruses
2) RNA Viruses
3) Reverse Transcription Viruses

Question 32

What are some DNA viruses?

Answer 32

ssDNA: Parvoviridae (linear), Circoviridae (circular), or Geminiviridae
dsDNA: Polyomaviridae (circular), Baculoviridae (circular), adenoviridae (linear, Herpesviridae (linear)

Question 33

What are some RNA viruses?

Answer 33

dsRNA: Reovirdaea, Birnaviridae, Partitiviridae, Chrysoviridae (there are not many)
+ssRNA: Picornaviridae, Togaviridae, Betaflexiviridae, Closteroviridae
-ssRNA: Orthomyxoviridae, Paramyxoviridae, Filoviridae, Bornaviridae

Question 34

What are some viruses involving reverse transcription?

Answer 34

Retroviridae: genome is RNA (integrate into host genome)
Caulimoviridae: genome is RNA (do not integrate into host genome)
Hepadnaviridae: genome is DNA (do not integrate into host genome)

Question 35

What are the cellular sites for each type of virus?

Answer 35

RNA viruses: Translated in cytoplasm, transcribed in cytoplasm, genome is replicated in cytoplasm
DNA viruses: Translated in cytoplasm, transcribed in nucleus, genome is replicated in nucleus
Retroviruses and para-retroviruses: Translated in cytoplasm, transcribed in nucleus, genome is replicated in nucleus

Question 36

What are some exceptions to the cellular sites for each type of virus?

Answer 36

Orthomyxo: they require nucleus for transcription and replication
Pox: they do everything in the cytoplasm of infected cell
NCLDV: require both nucleus and cytoplasm to complete replication

Question 37

What are Immediate early genes?

Answer 37

Genes expressed right after infection. Functions included:
1) Rendering cells enter S phase –> force cells to enter S phase
2) Induce expression of other viral genes
3) Inhibit host defence, and biosynthesis
Adenovirus bind to p53, block p53 mediated apoptosis and force cells to enter S phase/

Question 38

What are early genes?

Answer 38

Enzymes & accessory factors required for genome replication

Replication requires dNTPs, host machinery, strategies vary among viral families

Question 39

What are late genes?

Answer 39

Structural proteins required for assembly

Question 40

What are very late genes?

Answer 40

For some viruses, polyhedron baculoviruses

Question 41

How do DNA viruses work?

Answer 41

DNA viruses infect cells that are either actively diving or they force quiescent cells to enter S phase
DNA viruses do have the potential to be oncogenic
The only exception is Poxviridae, which replicate entirely in the cytoplasm and virions carry all enzymes & proteins required for transcription & replication

Question 42

What are some DNA viruses that have the potential to be oncogenic?

Answer 42

1) Adenoviruses subtypes A,C
2) Herpesviruses: Epstein-Barr virus (Burkitt’s Lymphoma)
3) Polyomaviruses: simian virus 40
4) Papillomaviruses: human papilloma virus type 16, 18

Question 43

How do RNA viruses work?

Answer 43

In general, all phases of the life cycle for RNA viruses occur in the cytoplasm
The exception is Orthomyxo & Borna
Viruses must associate with a type of intra-cellular membranes, these include:
1) Endoplasmic reticulum: TMV, potato virus X
2) Endosomal membrane: Sindbis virus (alpha, toga)
3) Vesicular membrane: Poliovirus
4) Peroxisome membrane: Tombusviridae
5) Mitochondria: Tymoviridae, Ampelovirus
6) Chloroplast: Tymoviridae

Question 44

How do positive stranded RNA viruses transcribe and translate?

Answer 44

Transcription & replication: RNA-dependent RNA polymerase (RdRP)
Genome Replication: Replicase enzyme
Transcription: Transcriptase enzyme
Positive strand RNA viruses: Genomic RNA serve as the 1st mRNA, and Replicase contains conserved domains included MTR, HEL, RdRP

Question 45

How do negative stranded RNA viruses transcribe and translate?

Answer 45

RNA genome exists as ribonucleoprotein (RNP) and not naked RNA complex, so transcribed before translation occurs

Question 46

How does the influenza virus transcribe and translate (negative stranded RNA)

Answer 46

1) RNPs enter the nucleus, where they replicate & transcribe
2) Splicing of transcripts made from some genome segments
3) NP contains NLS, also function as helices as it binds ssRNA

Question 47

What is special about retroviruses and para-retroviruses?

Answer 47

Defied the central dogma, meaning it went RNA –> DNA
These viruses have two identical +RNA molecules, RTase( virion: 50- 100 copies), integrase
Reverse transcription starts within virion upon entry into host cell
Integration of viral cDNA (provirus) into host chromosome, becoming part of host genome
Cellular DdRP II responsible for viral mRNAs and genomic RNAs

Question 48

How does assembly work in the viral replication cycle?

Answer 48

1) Formation of individual structural units of the protein shell from a single or a few structural proteins encoded by the virus
2) Assembly of protein shell by appropriate and sometimes variable interactions among structural units (large DNA viruses of eukaryotes and bacteria requires packaging protein to spool viral DNA into capsids
3) Selective packaging of viral genome and other essential virion components
4) Acquisition of an envelope (naked viruses lack this step)
5) Exit the infected cell
6) Maturation of virions (only applies to certain viruses, requires proteolytic cleavage, so naked viruses lack this step

Question 49

How does the assembly of structural units work?

Answer 49

The process of assembly to form structural units and virions is de novo: they do not need a template; spontaneous process due to interactions between capsid protein subunits
The interlocking of structural proteins is dictated by primary aa sequence (polypeptide)
Compartmentalization of viral replication and assembly in the cell
(There is a high concentration of proteins: to ensure assembly is correct, efficient, & directional
In the cytoplasm, all but one group of RNA viruses, and DNA viruses of Pox
In the nucleus, DNA viruses, retroviruses, orthomyxo

Question 50

How are assembly of structural units put together?

Answer 50

Image will be put in here

Question 51

How are capsid shells assembled for RNA viruses?

Answer 51

For RNA viruses, genome RNA is involved in virion assembly:
Tobacco Mosaic Virus and many other (+) ssRNA viruses
Poliovirus RNA may be involved in assembly and cleavage of virion, although not entirely know

Question 52

How are capsid shells assembled for DNA viruses?

Answer 52

In large DNA viruses and bacteriophages, procapsids are formed first, followed by collapse of scaffold protein structures, then genome DNA is inserted via portal of entry. This process requires input of energy by ATP hydrolysis

Question 53

How are the assembly of capsids and genome packaging done?

Answer 53

Look at TMV:
Structural unit: double disc 
Hairpin near 3' terminus of RNA genome forms the Origin of Assembly, double disc turns a 'lock washer'
Viral RNA threads through 
Building up of double discs

Question 54

What is the story of polioviruses?

Answer 54

There is an image to explain this, be sure to put in
Proteolytic cleavage of VP0 is the last step (maturation), renders the virion infectious
Proirion is not infectious until VP0 is cleaved into VP2 and VP4
Genomic RNA may be involved in the cleavage of VP0
Before VP0 is cleaved, virion is non infectious

Question 55

How does egress work in the viral replication cycle?

Answer 55

This occurs for naked viruses, and this is the lysis of infected cells:
–> Cytopathic effects (shutdown of host biosynthesis, destruction of cell structure, membrane alteration, chromosome breakup, syncytium, inclusion bodies)
For example, the adenovirus E1B disrupts the nuclear lamina and breaks done intermediate filaments, there is also destruction by attack by the host immune system

Question 56

Explain the assembly & egress of an enveloped virus?

Answer 56

Many animal and human viruses have envelopes as part of a virion
Source of envelope is determined by the glycoproteins of the virus
Budding and pinch off at the plasma membrane in most cases
Maybe put the picture in?

Question 57

What is the host range?

Answer 57

The range of hosts that can be infected by a given virus (natural host range vs experimental host range)
Natural: Very different
Experimental: Laboratory

Question 58

What is tissue tropism?

Answer 58

The preference of a given virus for certain types of cell and tissue in its hosts
Not all viruses will infect all cells and tissues
Viruses often prefer specific cells and tissues

Question 59

What are susceptible cells?

Answer 59

Cells that allow attachment & entry of a given virus due to presence of suitable receptor(s)
May or may not support viral replication

Question 60

What are permissive cells?

Answer 60

Cells that permit the replication of a given virus
These cells contain all factors required for viral replication
May or may not be susceptible
Permissive and susceptible cells are very different and have different meanings