2 - replication & structure (MICROM 450)

How does virus to cell attachment occur?

Virus attachment protein (VAP) binds to a receptor on cell surface (each virus species has a distinct receptor)

-Enveloped virus has glycoprotein(carb chain) spikes in membrane that act as VAP

-Non-enveloped virus use proteins on capsid

How do cellular receptors work?

-contain normal cellular molecules that the virus evolve to use

• ex. proteins, glycoproteins, carbohydrates, glycolipids

-binding leads to entry of virus into cells (multistep process)

-Receptor expression is a major determinant of TROPISM

What is Tropism?

Ability of a virus to productively infect a cell

-IF RECEPTOR NOT EXPRESSED ON CELL, VIRUS CANNOT ENTER

**not all viruses infect all cells

How do naked virus penetrate the cell?

Enter cell through endocytosis

-poke hole in host membrane & release genome in cytoplasm

• may release lytic protein to disrupt host membrane

Exit from endocytic vesicle is triggered by receptor binding or by changes in environment (low pH in endosome)

How do enveloped viruses penetrate the cell?

1. Fusion

   1. occurs at cytoplasmic membrane surface OR in endocytic vesicles AND require specific binding proteins to promote joining of the viral envelope with the host cell membrane

2. Endocytosis, then fusion

Positive (+) strand RNA

A type of RNA that serves directly as mRNA for protein synthesis in the host cell. It can be translated into proteins by the host's ribosomes without any additional processing.

How cand + strand RNA directly translate mRNA?

1. attachment to cell

2. penetration/fusion

3. uncoating (opening of protein capsid)

4. +, -, + (- to + will replicated 10xxxx)

5. assembly

6. maturation

7. release viral genome from cell

**ALL IN CYTOPLASM

In general, genomes are single polyproteins cleaved to make individual proteins. Why?

This mechanism is essentially a "one-message" system that serves several critical advantages for the virus, allowing it to function efficiently with limited genetic space

Viral replication of naked + strand viruses

1. attachment

2. endocytotic penetration

3. uncoating and translation of RNA

4. assembly of new virions

5. maturation

6. release from host cell

7. cell lysis to the extracellular environment (will burst out)

Negative (-) Strand RNA translation

1. attachment

   1. viral genome contains polymerase needed for - strand to transcribe a + strand necessary for translation

2. penetration

3. uncoating

4. -, +, - (translation of mRNA now possible)

5. assembly

6. maturation

7. release

DNA virus replication

1. attachment to cell

2. penetration to cytoplasm

3. uncoating (nucleus)

4. mRNA synthesis and translation into proteins

5. assembly of new virions

6. maturation (cytoplasm)

7. release from the host cell

In viral DNA replication, when do non structural proteins and structural proteins play a role/

• Non-structural proteins are involved in early replication processes such as genome replication, mRNA synthesis, and protein expression

• Structural proteins are synthesized later for the assembly of new virions/late gene expression

What do DNA viruses rely on to make viral mRNAs?

Host transcription complexes

-Ex. pox virus have their own transcription machinery to replicate in cytoplasm

Retrovirus replication

1. attachment to host cell receptors (polymerase intact)

2. penetration

3. uncoating / reverse transcription from RNA to DNA (nucleus)

4. integration into host genome

5. synthesis of viral proteins and RNA

6. assembly of new virions (leaves to cytoplasm)

7. maturation

8. release from the host cell

Retrovirus replication overview

Retrovirus replication involves virus attachment to host cell receptors, leading to penetration and uncoating. This is followed by reverse transcription where RNA is converted to DNA, integration into the host genome, synthesis of viral proteins and RNA, assembly, maturation, and finally release from the host cell.

What must retroviruses bring into the cell to the capsid to be able to replicate

reverse transcriptase enzyme

What occurs in the the Assembly step of replication?

Assembly is the building of the capsid & combining the nucleic acid w capsid to form new virions before maturation

• Virus particle can assemble around genome/ be put together separately & incorporate the genome after formation

• assembly occurs in nucleus or cytoplasm

What occurs in the the Maturation step of replication?

Maturation is stage of replication-cycle at which virus becomes infectious

• maturation involves structural changes in virus particle

• involves viral proteases & cellular enzymes

• can occur inside the cell or after release

What occurs in the the Release step of replication?

Release is the final stage of the viral replication cycle where new virions are expelled from the host cell. This can occur through cellular lysis or budding, allowing the virus to infect new cells.

How does cellular lysis release work?

Cellular lysis release occurs when the host cell is damaged or ruptured, causing the newly formed virions to spill out into the surrounding environment and infect nearby cells. This process results in the death of the host cell.

** mostly non-enveloped virus

How does cellular budding release work?

Cellular budding release is a process where new virions acquire their envelope by protruding through the host cell/cytoplasmic membrane. This method allows the viruses to exit while leaving the host cell intact, often permitting further viral replication.

** mostly enveloped virus

How does propagation of viruses occur?

• Obligate intracellular parasites are cultured in live cells

• Live animals (originally passaged in animals)

• Embryonated chicken eggs (viruses were cultivated on diff parts of the embryo)

• Cell culture

  • Primary cell culture: cells from og tissue

  • cell lines: cells can be induced to become immortal

  • cell culture: used to grow many animals viruses

• Virus plaques in culture to be quantified

What is Cytopathic effect? (C.P.E)

A change or abnormality in the microscopic appearance of cultured cells due to virus infection

What is Cytopathic effect cause by?

Interactions of virus and host cell caused by any stage of binding

**CPE can be cell death, cell fusion, and multi nucleation or cancerous cell growth

What is a Viral Plaque Assay?

A laboratory method used to quantify the number of infectious virus particles in a sample by counting the number of visible plaques formed in a cell culture.

**if viruses don’t form plaques, they don’t kill cells

How can we quantify viruses using plaque assays?

By counting visible plaques formed in cell cultures caused by infectious virus particles.

What are the steps for quantifying viruses?

1. make 10-fold dilutions of virus stock

2. use each dilution to infect a single layer of cells in a dish

3. overlay w media containing sugar & wait for plaques to form

4. stain cells & count number of plaques

5. back-calculating the pfu mL of stock gives accurate count of # of infections virus particles in your stock

What is PFU?

Plaque forming unit. It represents the ability of a particle to form a plaque in cell culture

**One infectious virus particle can cause one plaque

Do all virus particles each have PFU?

No, bc many virus particles are not infectious & will not lead to plaque formation

Happens when viruses don’t have a full genome, did not mature properly, VAP was destroyed somehow, mutations didn’t allow for full replication, etc

What are additional wats to measure/detect a virus?

• PCR: accurately measure # of particles by determining amount of viral nucleic acid

• Antigen tests: Eliza/rapid tests. Detect antibodies specific to viral protein to probe for presence of viral protein

• Serology: measures antibodies in the host's serum(blood) to indicate past exposure to the virus.

What are disadvantages to Serology?

1. Specific antibodies take over a week to appear after infection

2. cannot differentiate recent infection from very old to new infection

3. not quantitative

What are sizes of Virus particles?

nM = 10^-9, Angstrom = 10^-10

• Smallest thing seen by eye: 50,000 nM

• Human cell: 10,000-100,000 nM

• E coli: 500 × 1000 nM

• Range of virus particles: 20 nM to 350 nM

• Atomic radii: 0.1-0.3 nM OR 1-3 A

How can we determine the structure of viruses?

• e- microscopy

• x-ray crystallography

• cryo-EM

How can we use e- microscopy to determine virus structures?

Bombard sample w e- but need to create contrast btwn identical subunits

(50-70 angstroms)

How can we use x-ray crystallography to determine virus structures?

Make crystals and use xrays to hit crystals to measure defraction pattern to identify structure

• hard to get high quality crystals

(1-2 angstroms)

How can we use Cryo-EM to determine virus structures?

Freeze the sample in aq solution gives contrast to picture many particles and piece together to form 3D

(1.5 angstroms/ atomic level)

What are the steps to CryoEM Reconstruction?

1. Cryo-Plunge Samples

2. Collect Cryo-Electron Micrographs

3. Digitally Select Particle Images

4. 3D Image Processing

5. Structural Analysis & Processing

Why is knowing structure of a virus important?

Helps in ID of how virus particles interact w cells(receptors, glycoprotein spikes, etc), discovery of antivirals based on structure data(capsid interactions), understand stable structures, determines virus species(evolution)

Why do Virus structures need to be metastable?

1. must form tight impenetrable shell to protect nucleic acid

2. must break down once they enter a cell to allow uncoating of viral genome

3. should not cave cov bonds bc of need to break apart upon cell entry

4. maximal stability comes from contact btwn identical proteins

5. limited genome size means limited proteins, therefore repeating protein units in capsid is important

What are the 3 main eukaryotic Virus Structures?

1. Helical (Naked & Enveloped)

2. Icosahedral (Naked & Enveloped)

3. Complex (Enveloped)

What is helical symmetry?

• Rods/ cylindrical shaped

• all subunits made from single protein tightly packed brick by brick

• ALL helical viruses are enveloped

Ex. ebola

Why do people mistake helical for round capsids?

This confusion arises because some helical viruses can appear rounded or elongated due to their flexible structure, which may not exhibit a rigid cylindrical shape visually.

Icosahedral symmetry?

• shapes w 20 triangular faces (12 vertices)

• contain 3 diff axes of sym

  • 3-fold, 2-fold, 5-fold

What is the structure of isosahedral capsid pentamer look like?

5 proteins make up a penton (capsomers)

12 vertices (units)

5 × 12 = 60 identical subunits (protomers)

Pentons vs Hexons capsomers

• Pentons form pyramids for tighter binding, therefore form “vertices”

  • min of 5 protomers

• Hexons form flat “faces” when they come together

  • min of 6 protomers

What are the units that make up capsomers?

Protomers (minimum of 12)

ex. 5 protomers per penton x 12 vertices = 60 protomer units per capsomer

Icosahedral nucleocapsids

20 triangular face & 12 vertices (most efficient symmetric shell)

Examples of icosahedral capsids

1. small virus (parvoviruses) have 12 pentons/capsomers & no hexagonal capsomers

2. Larger viruses include more hexons enabling larger capsids to hold larger genomes

3. Herpesvirus have 12 pentons & 150 hexons (round look)

How can capsid subunits self assemble?

Made up of identical repeating units that interact via non-cov bonds which don’t require energy to make/break

• Some viruses express capsid protein alone will lead to self assembly virus like particle (VLP)