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Viral Pathogenesis > Exam 2- Basics of Infection > Flashcards

Exam 2- Basics of Infection Flashcards

1
Q

Susceptible cell

A

A cell that has a functional receptor for viral attachment and entry, but it may or may not support viral replication

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2
Q

Resistant cell

A

No receptor, may or may not support viral replication. Does not allow entry of the virus

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3
Q

What 2 characteristics must a cell have to allow viral uptake?

A

The cell must be susceptible and permissive to allow viral uptake and successful replication. For example, HIV replicates in CD4 T cells much better than it does in macrophages

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3
Q

Permissive cell

A

May allow replication of a virus, but may or may not be susceptible

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4
Q

How do we propagate viruses to study them in the lab?

A

The first method used was replicating the virus in chicken eggs- the virus grown using this method include influenza, HSV, mumps, and others

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5
Q

Immortalized cell lines

A

Used to culture viruses, these scientists won the Nobel prize in 1954. Cell lines used included primary human foreskin fibroblasts, mouse fibroblasts, and HeLa cells

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6
Q

How is infectivity measured?

A

Using the number of virus particles (viral titer)

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7
Q

Methods of measuring infectivity (6)

A
  1. Nucleic acid-based assays (RT-PCR, HTS)
  2. Plaque assay
  3. End-point dilution assay
  4. Serological assay (ELISA, immunostaining)
  5. Hemagglutination
  6. Electron microscopy/imaging
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8
Q

Plaque assay

A

Originally used to observe how bacteriophages infected bacteria, but they were adopted for mammalian cell culture. It is a measure of how many plaques are produced by viral infection on a culture dish. Plaques are a zone of clearance on the culture plate, because the cells have been infected and killed. More plaques demonstrate a higher level of infectivity or a higher concentration of the virus

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9
Q

Endpoint dilution assays

A

Based on cytopathic effects when viruses infect a cell- based on physiological and morphological effects. These assays quantify the viral titer in terms of tissue culture infectious dose. The cells are plated and diluted, and observed for a specific time period depending on the virus. If you see the morphological changes caused by the virus, the cell is scored as +, if not, the cell is scored as minus. The tissue culture infectious dose is determined based on the number of + wells. EDAs are not a very direct measure of virus titer

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10
Q

Electron microscopy

A

Imaging to count the number of virus particles

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11
Q

Cytopathic effects

A

Physiological and morphological changes when a virus infects a cell. The cell shape may change from its original structure. Some virus may cause cells to fuse together- like Koplik spots caused by measles

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12
Q

Tissue culture infectious dose (TCID)

A

The proportion of + wells in an end point dilution assay. + wells indicate an infected cell with morphological changes. If 50% of the wells are +, TCID is expressed as TCID50

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13
Q

How are plaque assays conducted if a virus doesn’t kill the cell?

A

You can engineer the virus to include beta-galactosidase as a fusion protein, which forms blue plaques.

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14
Q

How are the number of plaques in a plaque assay expressed?

A

In terms of plaque-forming units (PFU). PFU per mL is the best way to measure viral titer, and it gives you an idea of infectivity

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15
Q

Particle-to-PFU ratio

A

Every virus has a characteristic particle-to-PFU ratio that it produces in a plaque assay. Although a single particle can initiate an infection or form a plaque, not every particle is infectious- it could be damaged. For some viruses, you would need thousands of virus particles to cause one plaque. The lower the PFU, the higher the infectivity of the virus

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16
Q

Reverse transcription mechanism

A
  1. The RNA molecule extends its 3’ hydroxyl end using reverse transcriptase, which acts as a polymerase. tRNA acts as a primer
  2. A template transfer is necessary once there is no more RNA template to make additional DNA. There are R regions on DNA and RNA that are complementary to each other
  3. The 2 complementary regions anneal so the 3’ hydroxyl end can continue to expand
  4. As the DNA is synthesized, RNA is degraded
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17
Q

Polypurine track

A

The RNA sequence that cannot be degraded by reverse transcriptase. It acts as a primer for the synthesis of the second strand of DNA

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18
Q

RNase

A

Removes both RNA primers after reverse transcription

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19
Q

+ strand DNA synthesis

A

Each strand in the double helix acts as a template for synthesis of a new, complementary strand. New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5’ to 3’ direction.

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20
Q

Multiplicity of infection

A

The number of infectious particles per cell. For example, an MOI of 10 means that there are 10 virus particles per cell. The number can be misleading as the distribution is random (following Poisson distribution)- some cells may get a few virus particles while others might get none.

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21
Q

Importance of MOI

A

The MOI can change the response of cells to the infection, so knowing the MOI is important in understanding the kinetics of viral infection. In order to ensure that all cells are infected in the laboratory, you can use a higher MOI. This is called a one-step growth analysis

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22
Q

One-step growth analysis

A

After cells are infected with a virus, there will be an eclipse period, and then a sudden burst in the number of infectious virus particles. If only a fraction of the cells are infected, the viral particles produced by the first round of infectious cells will go on to infect the healthy cells. There will then be another cycle of the eclipse period and then the burst. Knowing the MOI is necessary for this analysis

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23
Q

Uses of a one-step growth analysis (4)

A
  1. Understanding the properties of viruses
  2. Understanding the infectivity of two different strains of a virus
  3. Understanding the kinetics of viral infection
  4. To test antiviral drugs- you should be using an MOI that’s higher than 1, so there’s only one step of growth
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24
During infection, how does the location of the virus change?
After the eclipse period, and until the latent period, the virus is intracellular. Beyond the latent period, the virus has shed into the extracellular environment
25
Hemagglutination
A phenomenon commonly observed in influenza, where red blood cells are agglutinized (form a cloud/lattice-like structure). Can occur in any virus with glycoproteins or fusion proteins on their cell surface. In wells, non-agglutinized cells form a "button" shape in the middle of the well, while agglutinized cells form a "cloud-like" appearance.
26
Immunostaining
Uses an antibody, conjugated with a fluorescent marker, against a particular viral protein or antigen. Then uses an immunofluorescent assay. If the antibody is able to bind to the antigen, the cell will appear fluorescent. The more binding that occurs, the brighter the fluorescence. Can be direct (the antibody binds directly to the antigen) or indirect
27
Indirect immunostaining
The primary antibody (without the fluorochrome) binds to the viral antigen. The secondary antibody (with the fluorochrome) binds to the primary antibody. It recognizes the Fc fragment of the primary antibody
28
Enzyme Linked Immunosorbent Assay (ELISA)
Similar to immunostaining, however, an enzyme is used to trigger a color change in the substrate rather than a fluorochrome. ELISA is able to detect either a viral protein (antigen) or virus specific antibodies. Can be direct or indirect (using 2 antibodies)
29
Viral antigen ELISA
Targets a captured antibody on the viral surface. The viral antigen is bound to the captured antibody, and a second antibody (bound to the indicator) binds the viral antigen
30
Viral antibody ELISA
An antibody in the sample (IgG) binds to the viral antigen on the cell surface. An anti-IgG antibody, bound to an indicator, is then used to bind to the initial antibody
31
Live-cell imaging
Live, single virus particles can be viewed through imaging. Fluorescent stains are used to target specific viral particles and cell structures
32
Polymerase chain reaction (PCR)
Uses Taq polymerase (for DNA) and reverse transcriptase (for RNA). DNA PCR or RT-PCR are frequently used to detect the viral genome and quantify the signal coming from the DNA/RNA component of the virus. A PCR product doesn't always mean the virus is infectious- a plaque assay is necessary for this
33
High throughput sequencing
If an unknown virus is present in a population, you can collect swabs and isolate the nucleic acid. Then, the sequence is compared with that of existing viruses and a phylogenetic tree can be developed. This is how novel CoV-2 was identified from a patient in Wuhan, China. Phylogenetic analysis of its RNA polymerase identified it as a member of the coronavirus family
34
How is ancestry determined using high throughput sequencing?
A root presumed ancestor is used to construct a phylogenetic tree. The distance between the common ancestor and the virus on the branch indicates the degree of genetic change, or how related the virus is to the ancestor
35
In which ways do viruses alter the cellular processes to facilitate their replication? (6)
1. Changes to the cell's genome 2. Changes to the transcriptome- gene expression 3. Changes to the proteome- proteins 4. Changes to the metabolome- glucose and lipids 5. Signal transduction- how cells transduce extracellular signaling downstream 6. Remodeling of cellular organelles, like the synthesis of membranes to help the virus assemble
36
Adenovirus PI3k signaling (5)
1. The penton fiber interacts with the fiber receptor and makes contact with the co-receptor (integrin) 2. Induces cell signaling that helps the virus to be internalized into the endosome through a coated pit 3. Adenovirus induces molecules such as Cas (protein), c-Src (kinase), and then PI3 kinase signaling 4. PI3k signaling remodels the actin filaments of the cytoskeleton under the coated pit. This is modulated by G proteins 5. The cytoskeleton must be rearranged, or else the endosome would not be able to cross
37
Influenza PI3k signaling (4)
1. The influenza virus engages with the sialic acid residue on the host cell surface 2. This promotes clustering (dimerization) of tyrosine kinase receptor proteins 3. Clustering promotes the receptors' kinase activity- they are autophosphorylated and PI3k signaling is activated 4. Actin network beneath the plasma membrane is remodeled, facilitating the entry of the endosome into the cell.
38
When are viruses able to modify cell signaling?
As soon as they engage with the host cell's receptor
39
Influenza Rptk signaling
1. Another kinase induced by PI3k 2. Activates Mapk 1/3 3. Map specifically activates the vesicular ATPase, which allows protons to enter the endosome. The pH drops 4. A cooperative effort between PI3k and Rptk allows the virus to uncoat itself
40
Viral proteins in the Akt signaling pathway (5)
1. Hepatitis C- non structuring protein 5A (ns5A) 2. Rotavirus- non structuring protein P1 3. Adenovirus- Ad5, E4, and Orf1. These proteins can act with PI3 kinase signaling 4. Hepatitis B- PI3k signaling 5. Herpesvirus- PI3k signaling
41
PI3 kinase structure
Composed of 2 subunits- P85 and P110
42
What is the purpose of the Atk pathway?
After infection, these proteins prevent the cell from dying. Cells will naturally undergo apoptosis if they can't fight off the infection. When Akt is activated, it promotes cell survival and activates apoptosis
43
mTor
The protein in the Akt pathway that is the master regulator of the protein synthesis process. Translation is the most abundant process occurring in the cell, and viruses need control of it so they can produce viral proteins
44
Herpes virus Atk pathway
Induces GPCR signaling, then going on to activate PI3k, and Akt, promoting cell survival. Cyclin D is also activated, promoting cell proliferation and autophagy. Finally, mTor is activated to take control of translation
45
Autophagy
A recycling process where cells can survive by recycling molecules for energy production
46
Why is inhibition of cellular gene expression necessary?
Viruses don't want to "share" cell machinery as they carry out their functions. As a result, they inhibit mRNA and protein production to inhibit cellular gene expression.
47
Mechanisms of cellular gene inhibition (3)
1. Some viruses will inhibit mRNA splicing, so mature mRNA can't be produced and it won't be translated. 2. Some viruses interfere with the export of DNA from the nucleus to the cytoplasm 3. Manipulate the stability of mRNA- mRNA has a very short half life and is already quickly degraded, but interfering with stability means that protein machinery may not recognize the mRNA. Viral mRNA is recognized instead
48
Viral nucleases
Responsible for degrading cellular RNAs to wipe out the competition. mRNAs are synthesized in the nucleus and shuttled into the cytoplasm, where they are bound by the necessary machinery, including proteins that bind to the mRNA cap and poly-A tail. The proteins make the mRNA into a circle, and it is translated in a circle as well. Viral nucleases function as endonucleases- they cleave the mRNA into pieces, preventing it from being bound to any proteins. Then the mRNA is vulnerable to cellular exonucleases, it will not be translated
49
Examples of viral nucleases (4)
1. Influenza A- PA-X 2. SARS- NSP1 3. KSHV- SOX 4. VHS- HHV-1
50
Exonucleases
Constantly degrade cellular mRNA. These enzymes are what determine the half-life of mRNA in the cytoplasm. An example of these enzymes is Xrn-1
51
Phases of protein synthesis in a virally infected cell (3)
1. All cellular mRNAs are blocked from beginning protein synthesis, host cell protein synthesis decreases (hour 0-2.5) 2. Protein synthesis occurs, but only for viral mRNA (hour 2.5-4) 3. Viral protein synthesis decreases (4+)
52
Viral proteins that inhibit host protein synthesis (3)
1. eIF4E binds cap on host mRNA 2. eIF4E- helicase activity- results complex secondary and tertiary structures in mRNA so ribosomes can recognize codons 3. eIF1A- AUG recognition protein (stop codon)
53
Internal Ribosome Entry Sites (IRES)
Found in the 5' end of the genome of some viruses. Viruses encode a mechanism where viral mRNAs can be recognized by cellular machinery, independent of cap recognition. All of the sites form different structures and are directly recognized by cellular translation machinery. They are meant to direct ribosomes to the correct site. IRES is not a conserved sequence
54
Cap-independent translation initiation
Initiated by IRES sequences. This allows for necessary subunits, including the small subunit of the ribosome, to translate the mRNA and recruit other necessary factors. The C terminal of eIF4G and eIG3 recruit the 40s (small) ribosome subunit
55
Hepatitis C IRES
Only eIF2 and eIF3 are required to carry out cap-independent translation initiation
56
Application of IRES discovery
Bacteria has polycistronic DNA that makes polycistronic mRNA. One gene can be used to transcribe multiple types of proteins. Eukaryotic cells are the opposite, they only produce one protein. However, to produce two proteins from a gene, two genes can be separated using an IRES sequence. Ribosomes can bind to each region separately and make 2 different proteins. This technique can be used to clone a gene
57
Regulation of translation initiation (3 steps)
1. After recognition of the Cap-binding machinery, initiation factor (eIF2) is recruited 2. Kinases phosphorylate eIF2 in case of stress in the cell, stopping protein synthesis 3. eIF2 binds to eIF2B, forming a stable complex. eIF2 is then presented in rate-limiting amounts. If the amount of free eIF2B declines, translation initiation is inhibited
58
eIF2B
eIF2 is recycled by eIF2B for each new synthesis of the polypeptide chain
59
Gcn2
A kinase that phosphorylates eIF2. Pauses translation initiation in response to amino acid starvation
60
Protein kinase R (Pkr)
Specifically activated in the presence of double stranded RNA in the cytoplasm. dsRNA is abnormal, it will only be present with a viral infection. When activated, Pkr phosphorylates eIF2 to shut off protein synthesis
61
Cell response to activation of Pkr
When Pkr finds dsRNA in the cytoplasm, it dimerizes and the proteins phosphorylate each other. The phosphate is transferred to eIF2. eIF2 sequesters eIF2B to pause protein synthesis
62
Pkr function
Has 2 domains- a kinase domain and a domain that recognizes double stranded RNA.
63
Viral mechanisms that counter inactivation of eIF2 (4)
1. K3L (vaccinia) and gamma-34.5 (HSV) act as phosphatases- phosphate is removed from eIF2 so it will sequester eIF2B 2. Inhibit the activation of endoplasmic reticulum kinase (Perk) 3. Inhibit the binding of Pkr to double stranded RNA so the dsRNA cannot be recognized 4. Transcribing antagonistic RNA from the viral genome
64
What do viruses need? (2)
1. Building blocks- nucleotides, amino acids, and fatty acids 2. Energy- 4 ATPs are required to make one peptide bond
65
Which biosynthetic processes do viruses manipulate?
All biosynthetic process, including glucose and lipid metabolism
66
Glucose metabolism yields
The glucose to pyruvate conversion (glycolysis) yields 2 ATPs and 2 NADH. Glycolysis can be shunted into 2 other pathways- the pentose phosphate pathway and the Krebs cycle
67
Pentose phosphate pathway
Responsible for nucleotide synthesis. Produces ribose 5-phosphate, needed for synthesis of dNTP
68
Acetyl-CoA
The precursor for fatty acid synthesis
69
Increased glucose consumption during viral infection
Dropping pH causes the increased consumption of glucose, producing lactate (an acid). Hepatotropic viruses (e.g. HBV, HCV) cause Type 2 Diabetes by altering the enzymes involved in glucose synthesis
70
Kinases
Add a phosphate group onto a substrate
71
Triacylglycerol structure
3 fatty acids complexed with a glycerol molecule. This is how lipids exist in our body.
72
Lipoprotein
Triacylglycerol can be complexed with proteins to create a lipoprotein, which is trafficked in the blood circulation. Cells metabolize lipoproteins using membrane associated LPL (lipoprotein lipase) enzymes, converting it into free fatty acids. The free fatty acids can be taken up by cells through the designated channels
73
Acyl-CoA synthase
Acts on free fatty acids to make acyl-CoA, which is used to produce energy. Acyl-CoA can be brought to the mitochondria or the endoplasmic reticulum
74
Triacyl glycerol
Acyl-CoA is converted to triacyl glycerol in the endoplasmic reticulum. Can be used in the body or to form a perilipin protein coat
75
Perilipin protein coat
Produced through processing of triacyl glycerol. When it is released from the ER, TAG complexes with proteins. Multiple lipid droplets coalesce together to form the protein coat, which is a double membrane vesicle. Viruses can override normal lipid metabolism to produce the coat
76
HCMV and fatty acid biosynthesis
77
Srebp1
A transcription factor that needs to go inside the nucleus in order to induce the expression of genes that carry out the fatty acid biosynthetic pathway. When translated in the ER, Srebp1 is produced as a precursor and is resident in the ER membrane. Regulation of Srebp1 production is due to cholesterol in the cell
78
Regulation of Srebp1
When there is enough cholesterol in the cell, Srebp1 is bound to SCAP and INSIG1, keeping it sequestered in the ER. If cholesterol levels drop, Srebp1 is released from the ER. In a vesicle form, it travels to the Golgi apparatus, where it is cleaved by Golgi resident peptidase into a mature form called Srebp1-M. The mature form enters the nucleus and acts as a transcription factor, influencing the expression of genes that act in fatty acid metabolism
79
HCMV and Srebp1
HCMV disrupts the maturation process of Srebp1. Independent of the presence of cholesterol, HCMV can induce the maturation of this transcription factor. It does this by inducing the activity of ER resident kinase (Perk), which phosphorylates eIF2. When active, Perk inhibits INSIG1, so INSIG1 can't keep Srebp1 sequestered in the membrane. Srebp1 goes to the Golgi, matures, then goes to the nucleus as a transcription factor
80
mTORC1
Found in HCMV. Involved in promoting proliferation and cell growth. It can inhibit Lipin-1, which inhibits the entry of SREBP1 into the nucleus
81
Why do some viruses promote fatty acid metabolism?
Lipid droplets are like sponges for protein. Viral proteins that are produced will remain in the same area as the fatty acids

Viral Pathogenesis (11 decks)

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