M8 - virus

learning outcomes

• explain why viruses are obligate, intracellular parasites

• describe how viruses are different from other obligate intracellular parasites

• identify parts of virion and describe their function

• distinguish enveloped viruses from nonenveloped viruses

• describe the five steps common to the life cycles of viruses

• describe the two most common methods for virion release from a host cell

• compare and contrast the major steps of the life cycles of virulent phages and temperate phages

• list the approaches used to cultivate viruses

• describe direct and indirect counting methods used to enumerate viruses

• identify similarities and differences between the 7 groups of viruses discussed in lecture

• describe applications of viral based vectors in gene therapy

Explain why viruses are obligate, intracellular parasites

viruses: acellular entities that are only able to reproduce within living cells

viruses do not have:

• enzyme systems to produce basic chemical building blocks of life

• enzyme systems to produce ATP

• ribosomes, tRNAs and enzymatic machinery to direct protein synthesis

viruses must be able to

• induce changes in host cells → viral genes are replicated and viral proteins expressed

• use host cell equipment for replication of viral nucleic acids

• use host cell protein synthesis machinery + metabolic energy resources

Describe how viruses are different from other obligate intracellular parasites

Identify parts of virion + describe function

virion: a complete virus particle

• has a range in size

• must be visualized w electron microscope

composition of virus

• simplest virion: nucleocapsid

  • nucleocapsid: nucleic acid + protein coat

    • nucleic acid can be DNA or RNA

    • protein coat: capsid

      • protects viral genome

      • aids in transfer between host cells

      • encoded by viral genome

      • capsid proteins: protomers

• enveloped viruses: nucleic acid + capsid + lipid bilayer

  • lipid bilayer/envelope has spike proteins

    • spike proteins: embedded viral glycoproteins

capsid morphologies

1. spherical or icosahedral or polyhedral

2. rod-shaped or helical

3. complex

viral genomes = structurally diverse

• viruses are the only forms of “life'“ w RNA genomes

• DNA viruses share gene expresson/replication patterns w host cell

• RNA viruses have to find a way to replicate genome and synthesize mRNA

  • has specific enzymes not in host cell

    • RNA dependent RNA polymerase

Describe 5 steps common to virus life cycles + 2 most common virion exits

1. attachment or adsorption

2. host cell entry

3. synthesis or viral replication

4. viral particle assembly

5. exit/egress of virion

Attachment or adsorption

1. virion-cell surface receptor interaction

2. entry

• receptor determines tissue tropism + host range of virus

  • tissue tropism: the ability of a given pathogen to infect a specific organ or sets of organs

  • host range: the diversity of species that viruses can naturally infect

• virus takes advantage of host receptors that are always present

  • always present = vital for normal cell function

  • ensures infection

• enveloped viruses have glycoproteins that serve as binding sites

• non-enveloped viruses have protrusions/cavities that bind to surface proteins on host cell

host cell entry

• involve receptor-mediated endocytosis (cellbi reference !)

• enveloped viruses enter via 2 mechanisms

  1. fusion w PM

  2. endocytosis followed by fusion w endosome membrane

• non-enveloped viruses enter via translocation

• bacteriophages insert nucleic acid into cytoplasm, capsid left outside

synthesis or viral replication

• transcription and translation of viral genes

• RNA viruses require RNA dependent RNA polymerase

• DNA viruses use host’s DNA replication + mechanisms

  • may encode their own DNA polymerase or use the host cells

within host cell, virus requires

• viral replication enzymes = viral polymerases

• transcription factors for gene expression of other viral gene products

• structural proteins = capsid proteins + surface glycoproteins

not all viral genes = expressed at once

• temporal regulation of gene expression

• needed early:

• needed later:

viral particle assembly

• viral gene expression + viral genome production → assembly of new viral particles

• different for helical and icosahedral capsids

  • self-assembly

exit of virion

1. lysing host cell

2. budding

• lysing is common for bacterial viruses + non-enveloped animal viruses

• budding is observed for enveloped viruses

  • host cell may survive + continue releasing virions

  • all envelopes from host cell membrane

Compare and contrast the major steps of the life cycles of virulent phages and temperate phages

List approaches used to cultivate viruses

1. Lytic and archaeal viruses

   • mix cells and virus in broth culture → visions released into broth

2. Temperate viruses

   • cultured w cells on solid medium → lysis observed as plaques

3. Animal viruses

   • inoculated into suitable animal host ? ASK

Describe direct and indirect methods of enumeration of viruses

Direct

• electron microscopy

• Epifluorescence microscopy

• Q PCR

Indirect

• hemagglutination assay

• plaque assay

• Determining lethal dose LD50

Hemagglutination assay

• many animal virus particles bind to RBC surface

• if ratio virions:cells is large enough → virions will join the RBCs

• the more agglutination the more virions are present

plaque assay

• several dilutions of sample containing virus particles plated with appropriate host

• plaque count can be used to estimate number of virions in OG sample

• expressed as PFU: plaque-forming units BC

  • not all virions may be infective

  • possible for more than one virion to infect same cell

  • two infected cells to be plated on same area → single plaque

lethal dose or infectious dose

• used when biological effects not readily quantifiable with hemagglutination assay or plaque assay

• organisms/cell cultures inoculated w serial dilutions of a preparation of virus particles

lethal dose and infectious dose

• LD50 = dilution that contains a concentration of virions large enough to destroy 50% of the host cells or organisms

• ID50 = the dose that causes 50% of host organisms to become infected

identify similarities and differences between the 7 groups of viruses discussed in lecture

plus strand vs minus strand

• compares RNA genome to mRNA produced

• (+) ssRNA viruses: RNA genome identical in base sequence of the mRNA they produce

• (-) ssRNA viruses: RNA genome complementary to the mRNA they produce

double stranded vs single stranded DNA viruses

dsRNA(-/+)

1. RNA → DNA via their own RNA synthesizing enzyme (RdRp)

2. one strand of dsRNA → ssRNA(+) via RdRp transcriptase activity

3. ssRNA(+) released into host cytoplasm

4. ssRNA(+) translated by host cell machinery

• ssRNA(+) can act as template for (-) RNA strand synthesis that is converted back into dsRNA genome for new viral particle synthesis

ssRNA(+)

1. ssRNA(+) can be directly translated into viral proteins

2. RdRp translated first

3. RdRp copies ssRNA(+) into complementary (-)

4. ssRNA(-) can act as template for new (+) strand synthesis

5. new (+) strands are packaged and released

ssRNA(-)

1. genome cannot function as mRNA

2. has at least one RdRp protein into host during entry

3. ssRNA(-) serves as template for ssRNA(+) strand synthesis

4. ssRNA(+) act as templates for new (-) genomes

ssRNA-RT (retroviruses)

1. ssRNA(+) aren’t translated

2. uses reverse transcriptase to make DNA copy of RNA

3. viral DNA is then integrated into a host cell chromosome as a provirus

4. provirus can remain latent or be expressed

dsRNA-RT (reverse transcribing DNA viruses)

1. use reverse transcriptase and RNA intermediate to replicate genome

2. has a circular dsDNA molecule that consists of one complete but nicked and complementary strand with a large gap

3. viral gapped DNA released into nucleus

4. host repair enzymes fix the gap and the nick → covalently closed circular DNA (cccDNA)

5. transcription cccDNA yields mRNA → translated of mRNA → viral proteins