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MICR 3330 Midterm > Lecture 4 and 5 > Flashcards

Lecture 4 and 5 Flashcards

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1
Q

What did Enders, Weller and Robbins do in 1942?

A

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

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2
Q

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

A

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

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3
Q

What are cell cultures used for?

A

Widely used in research and vaccine development

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4
Q

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

A

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

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5
Q

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

A

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

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6
Q

What are Cytopathic effects, or CPE?

A

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)

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7
Q

What are the differences between an animal and plant cells?

A

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

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8
Q

Explain the One-step growth cycle

A

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

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9
Q

Explain the parts of the one step growth cycle

A

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

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10
Q

What is the MOI?

A

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

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11
Q

What is attachment in the virus replication cycle?

A

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

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12
Q

What are the interactions that occur in the attachment phase?

A

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

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13
Q

Explain the plasma membrane at the surface of eukaryotic cells

A

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

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14
Q

Where are membrane proteins are attached?

A

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

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15
Q

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

A

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’

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16
Q

What are some cell receptors for naked viruses?

A

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

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17
Q

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

A

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

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18
Q

Explain receptors

A

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

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19
Q

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

A

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

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20
Q

What is the model for poliovirus entry?

A

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

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21
Q

How does the model for poliovirus entry work?

A

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

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22
Q

Explain the entry & un-coating at the plasma membrane

A

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

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23
Q

What is the cellular function of receptor-mediated endocytosis ?

A

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

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24
Q

How does receptor mediated endocytosis work?

A

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

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25
How does receptor mediated endocytosis & uncoating @ endosomal membrane work for influenza?
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
26
How does receptor mediated endocytosis & uncoating @ endosomal membrane work for adenoviruses (naked viruses)?
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
27
How do phages with dsDNA genomes: T4 enter the cell?
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
28
What are the routes of entry of plant viruses into plant and plant cells?
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
29
What are some important terms for the biosynthesis of the viral replication cycle?
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)
30
What is the revised Baltimore system
Put in picture
31
What are the three broad categories of viruses based on genomes?
1) DNA Viruses 2) RNA Viruses 3) Reverse Transcription Viruses
32
What are some DNA viruses?
ssDNA: Parvoviridae (linear), Circoviridae (circular), or Geminiviridae dsDNA: Polyomaviridae (circular), Baculoviridae (circular), adenoviridae (linear, Herpesviridae (linear)
33
What are some RNA viruses?
dsRNA: Reovirdaea, Birnaviridae, Partitiviridae, Chrysoviridae (there are not many) +ssRNA: Picornaviridae, Togaviridae, Betaflexiviridae, Closteroviridae -ssRNA: Orthomyxoviridae, Paramyxoviridae, Filoviridae, Bornaviridae
34
What are some viruses involving reverse transcription?
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)
35
What are the cellular sites for each type of virus?
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
36
What are some exceptions to the cellular sites for each type of virus?
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
37
What are Immediate early genes?
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/
38
What are early genes?
Enzymes & accessory factors required for genome replication | Replication requires dNTPs, host machinery, strategies vary among viral families
39
What are late genes?
Structural proteins required for assembly
40
What are very late genes?
For some viruses, polyhedron baculoviruses
41
How do DNA viruses work?
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
42
What are some DNA viruses that have the potential to be oncogenic?
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
43
How do RNA viruses work?
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
44
How do positive stranded RNA viruses transcribe and translate?
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
45
How do negative stranded RNA viruses transcribe and translate?
RNA genome exists as ribonucleoprotein (RNP) and not naked RNA complex, so transcribed before translation occurs
46
How does the influenza virus transcribe and translate (negative stranded RNA)
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
47
What is special about retroviruses and para-retroviruses?
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
48
How does assembly work in the viral replication cycle?
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
49
How does the assembly of structural units work?
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
50
How are assembly of structural units put together?
Image will be put in here
51
How are capsid shells assembled for RNA viruses?
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
52
How are capsid shells assembled for DNA viruses?
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
53
How are the assembly of capsids and genome packaging done?
``` 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 ```
54
What is the story of polioviruses?
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
55
How does egress work in the viral replication cycle?
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
56
Explain the assembly & egress of an enveloped virus?
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?
57
What is the host range?
The range of hosts that can be infected by a given virus (natural host range vs experimental host range) Natural: Very different Experimental: Laboratory
58
What is tissue tropism?
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
59
What are susceptible cells?
Cells that allow attachment & entry of a given virus due to presence of suitable receptor(s) May or may not support viral replication
60
What are permissive cells?
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

MICR 3330 Midterm (8 decks)

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