Summary of the last lecture
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Summary of the last lecture. Virus classification schemes: Classical Recent: ICVT Baltimore Virus Structure: Virion DNA or RNA, virus replication enzymes Capsid, or nucleocapsid Icosahedral and helical Envelope(lipids and carbohydrates from cells) Glycoproteins (Gp, G): spikes

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Summary of the last lecture

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Summary of the last lecture

Summary of the last lecture

  • Virus classification schemes:

    • Classical

    • Recent: ICVT

    • Baltimore

  • Virus Structure: Virion

    • DNA or RNA, virus replication enzymes

    • Capsid, or nucleocapsid

      • Icosahedral and helical

    • Envelope(lipids and carbohydrates from cells)

    • Glycoproteins (Gp, G): spikes

    • Matrix protein

    • Assessory proteins

  • Virus assembly

    • Spontaneous

    • Protein-protein, protein nucleic acid and protein-lipid interaction (hydrophobic and electrostatic)


Summary of the last lecture

Assembly of poliovirus


Summary of the last lecture

Mechanism of packaging genome

  • Direct contact of genome with nucleocapsid or capsid

    proteins: small viruses such as poliovirus contain

    specific proteins that interact with RNA.

  • Specialized viral encoded nucleic acid binding proteins:

    these proteins, called ribonucleoproteins, nucleoproteins

    or core proteins, contain a nucleic acid binding motif in

    the sequence, or recognize a specific packaging signal

    sequence in RNA or DNA. These proteins are highly basic

    which react with negatively charged nucleic acids.

  • Packaging by cellular proteins: unique to papovaviruses, so

    the small viral genome doesn’t need to devote its limited

    genome to packaging.


Packaging sequence in herpesvirus

Packaging sequence in Herpesvirus


Cell culture

Cell culture

  • Medium for cell growth

    • -- water, salts, minerals

    • -- amino acids

    • -- glucose

    • -- serum

    • -- antibiotics (penicillin and streptomycin)

    • -- special growth factors (for some cell types)

  • Cell culture

    • -- primary cell culture: prepared freshly from tissue

    • -- cell lines (immortalized cell culture):directly derived from tumor or primary cells transformed with tumor virus or chemical mutagen


Cell culture cont

Cell culture (cont’)

  • CO2 incubator: temperature 37oC, humidified, co2

  • Long term storage: at liquid nitrogen (-140oC). Cells should be frozen quickly in -80oC freezer and thaw rapidly at 37oC waterbath


Primary cells vs cell line

Primary cells vs. cell line


Cell types

Cell types

Fibroblast-like cells (BHK)

Epithelial-like cells

(Lung carcinoma)


Cytopathic effects cpe

Cytopathic effects (CPE)

uninfected

0.01PFU of virus/cell infected

0.1PFU of virus/cell infected

10 PFU of virus/cell infected


Cpe cont

CPE(cont’)

Intranuclear inclusion body(LM)

Syncytium

Intranuclear inclusion body(EM)


Virus cell interaction

Virus-cell interaction

  • Attachment (reversible)

    -- virus attachment proteins (capsids, or glycoproteins)

    -- receptors or co-receptors (cell surface molecules)

    -- virus tropism ( spectrum of cells that virus can infect)

  • 2. Entry (penetration cross the plasma membrane, irreversible)

    • -- Receptor mediated endocytosis (the most common)

    • -- Cell membrane fusion (usually enveloped virus)

    • -- Translocation: very rare

  • 3. Uncoating( release of nucleic acid from its protein coat)

    • -- uncoating at the plasma membrane (Paramyxovirus)

    • -- uncoating triggered by change in pH (Influenza)

    • -- uncoating in the cytoplama


Virus attachment

Virus attachment


Viral receptors

Viral receptors


Virus entry

Virus entry


Virus entry cont

Virus entry (cont’)


Virus entry inhibitors

Virus entry inhibitors


Virus cell interaction cont

Virus-cell interaction (cont’)

  • 4. Replication and protein synthesis

    • -- In general, early proteins ( viral enzymes, regulatory

    • proteins) are synthesized first, followed by virus

    • genome replication and late proteins (structural

    • proteins) synthesis

  • 5. Assembly and release

    • -- Non-enveloped virus become mature inside the cell

    • and are released by cell lysis

    • -- enveloped viruses mature by budding from the plasma

    • membrane of host cells


Virus release

Virus release


Virus budding

Virus budding


One step growth curve

One-step growth curve

Pfu/cell(log)


Applications of cell culture

Applications of cell culture


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