Virology Exam I

Virus

consist of a nucleic acid genome (DNA or RNA) packaged in a protein coat. Viruses are either RNA or DNA, not both.

Capsid

protein coat that packages a virus

Virion

the complete, infectious virus particle

Size of Viruses

unit of measurement is in nanometers (nm), they range from 20-150 nm in diameter, larger virus are more complex and have larger genomes that code for more proteins

parvovirus (-18nm) more dependent on cell host

poxivirus (-300nm)

Virus depend on..

living cells for their replication - obligatory intracellular “parasites”

because they need cellular machinery (ex. enzyme system and others) for their genome replication and protein synthesis

Why study viruses?

1. viruses are important disease-causing agents

2. viruses can infect all forms of life

3. viruses are the most abundant form of “life” on Earth

4. the study of viruses has led to numerous discoveries in molecular and cell biology

Filterable Viruses

early experiments demonstrated that the mysterious agents causing diseases like tobacco mosaic disease, foot-and-mouth disease in cattle, and yellow fever in humans could pass through fine filters designed to hold back bacteria.

origin of virus- latin word “posion”

Viruses are significantly smaller than bacteria, which allows them to easily pass through bacterial filters.

Detection and Titration of Viruses

1. Bacteriophage plague forming assay

2. Eukaryotic cells for plague assays to measure Plaque-forming Units (PFU)

3. Hemagglutination assay

Bacteriophage plaque forming assay

1. Serially dilute virus (bacteriophage) stock;

2. Mix diluted viruses with cultured bacteria in agar-containing media and pour

into Petri dishes;

3. Grow the bacteria and viruses and allow plaques to develop

Eukaryotic cells for plague assays to measure plague-forming units (PFU)

1. Plate cells with 90-100% confluent (coverage);

2. Serially diluted virus samples add to the cells;

3. Cover the cells with agarose containing medium;

4. Grow the cells and viruses for several days and stain the plates.

5. Count plaques

Hemagglutination assay

Many animal viruses bind to sialic acid residues or other

carboyhydrates on cell surface proteins and lipids.

Red blood cells have carbohydrate-containing receptors (receptors)

on their surface.

Virus particles have multiple copies of receptor-binding proteins

(ligands) on their surface, and red blood cells contain many copies

of surface receptors.

Forms an interlaced network of adjacent cells.

In contract, without presence of viruses individual red blood cells

slide to the bottom of the tube and form a compact, dark red

pellet.

Physical Viruses v.s. Infectious Particles

the ratio of physical particles to infectious particles can be 10,100, even 1000

not all virus particles may be intact

some virus particles may contain defective genomes

“empty“ capsids (coats) that contain no viral genome can be made in large numbers

the virus replication cycle

1. Binding to cell receptor

2. entry and uncoating

3. early gene expression

4. replication of viral genome

5. late gene expression

6. assembly of virions

7. exit

Multiplicity of Infection (moi): this is defined as the number of infectious virus particles added per susceptible cell

Basic concepts of virus structure

a cardboard is rigid, symmetrical, and fixed in volume and shape

• Withstand the extracellular environment;

• Attach to and enter host cells, and release the viral genome into the

the host cell.

Naked or Enveloped

virus with no envelope is a naked virus (ex. adenovirus and infectious canine hepatitis virus)

virus with an additional lipid bilayer membrane wrapped around the capsid of the virus particle is an enveloped virus (ex. influenza virus and west nile virus)

Virus structure is studied by….

1. Electron Mircoscopy: high resolution revelas the general outlines of a vitus particle, and shows the bumps and projections formed by oligomers of viral structural protiens on the surface of the virion

• Cryoelectron miscroscopy: samples are flash-frozen in liquid nitrogen and kept at that temp in the microscope (improves preservatio of structures but image contrast is low)

• computer builds the virion structure

2. X-ray diffraction: virion crystallization; greater structure resolution than electron microscopy, allow precise determination of the position of every atom in the virion.

• xray cannot be focused - computer build 3D structure

• many enveloped virus cannot form crystals - individual membrance proteins can be purified and crystallized, allow determination of their molecular structures

Negative and Positive Staining

negative: reveals structure of virion by the exclusion of electron absorbing molecules such as uranyl acetate (background darker than virus particle)

positive: for proteins, nucleic acid, and lipids (virus particle darker than background)

Virus Structure

capsomer: a basic subunit of the capsid proteins (repeating protein subunits)

• arranged symmetrically around the viral genome

• very stable

Viral Shapes

Icosahedral: has 20 triangular faces regularly

Helical: a closed shell for packaging viral genomes in an elongated tube

Complex viruses don’t fit either because they are larger and have a more complex genome

Viral envelopes are made from

host lipid bilayer membranes! enveloped viruses acquire this membrane primarily through budding, where the nucleocapsid pushes through the host cell membrane.

• viral glycoproteins inserted into the host membrane and become embedded to the outer surface of the released virion

• host cell membrane proteins are present along the modified bilayer

packaging of genomes and virion assembly

• capsid assembly: the structural proteins assemble to form the protective viral capsid

• specifc packaging signals direct the selective incorporation of viral genomes into the virions. core proteins may accompany the genome inside the capsid.

• envelope acquisiton: the formation of viral envelopes occurs via the budding process as the nucleocapsid exits through the host membrane

Viral Classification

• host organism

• virus particle morphology

• genome type

• viral characteristics and structure

• diseases they cause

• geographic location of their discovery

• tissue/body site of initial isolation

• means of transmission

The seven groups in the Baltimore classification (genome) system

1. +ssRNA

2. -ssRNA

3. dsRNA

4. dsDNA

5. ssDNA

6. +ssRNA

7. ss/dsDNA

Major Virus groups based on Baltimore classification

• Viruses with single-stranded DNA genomes

• Viruses with double-stranded DNA genomes

• Viruses with double-single-stranded DNA genomes

• Viruses with positive-strand RNA genomes

• Viruses with negative-strand RNA genomes

• Viruses with double-stranded RNA genomes

• Viruses that use a reverse transcriptase

Satellite viruses, satellite nucleic acids, and viroids

Satellite viruses: defective viruses that require a helper virus to replicate

Satellite nucleic acids: small nucleic acid molecules (DNA or RNA) that depend on a helper virus for replication

Atypical Virus-like Agents

Defective Viruses: complete virion but unable to replicate without helper virus

Pseudovirions: contain host DNA not viral, infectious but no replication

Viroids: small circular RNA molecules; no capsid or envelope

Prions: infectious proteins with no detectable nucleic acids

• cause transmittable spongiform encephalopathies

Key steps in virus life cycle

1. attachment

2. penetration (entry) and un-coating

3. viral genomes replication

4. assembly

5. maturation

6. release

Attachment

the first step to establish a viral infection

• cellular surface receptor dependent: receptors are usually critical for cell survival; some viruses use primary and secondary (co-receptors)

Penetration (entry) and Un-coating

Enveloped Viruses:

1. Fusion with the cell membrane

• result of receptor-mediated fusion (pH independent)

• enveloped viruses that contain fusion proteins

• nucleocapsid is released directly into the cytoplasm

2. Fusion with an endosome membrane

• result of receptor-mediated endocytosis (ph dependent)

• involves binding to clathrin-coated pits on the host cell surface

• release into the cytoplasm is stimulated by the low pH of the endosome

Naked Virus

3. Endocytosis of naked viruses

• uncoating within the endosome

Viral genome replication: types of viral genomes and their replication

1. The production of viral structural proteins and enzymes

2. Replication of the viral genome

What three enzymatic activities does reverse transcriptase (RT) possess?

1. RNA dependent DNA polymerase: synthesizes single stranded cDNA (-) using the viral +ssRNA genome as a template

2. RNase H activity: degrades/cleaves the original RNA template from the resulting RNA/DNA hybrid

3. DNA-dependent DNA polymerase: synthesizes the complimentary DNA strand (+), creating double-stranded DNA

DNA Viruses

Most DNA target the nucleus

• can use the cell’s DNA-dependent RNA polymerase II for mRNA production

• Can also use the cell’s processing enzymes

Except for the viruses that replicate in the cytoplasm (poxviruses) must provide these functions themselves (packaged within the virion - large genome)

RNA Viruses

Most RNA viruses replicate and produce mRNA in the cytoplasm

• +ssRNA genomes can be directly translated into viral proteins

• other types of RNA virus: transcribe into mRNA before translation

• because host cells don’t know how to replicate RNA, all the RNA virus must carry an RNA-dependent RNA polymerase that will synthesize the viral +ssRNA, mRNA, and -ssRNA

(ss or ds, + or - sense, linear or segmented)

Retroviruses

viruses with ssRNA genomes that use a dsDNA intermediate to replicate

• uses RNA as its genetic material and truns it into DNA inside a host cell using a special enzyme called reverse transcriptase

Assembly

Polypeptide precursor accumulation along the inner host cell membrane alongside viral RNA

envelope protein association: transmembrane viral envelope proteins and surface glycoproteins anchor in the host cell membrane

budding: the viral genome and precursor proteins push outward through the host membrane, forming an immature virion that pinches off

Viron Maturation and Viron Release

lytic viruses: most naked viruses

latent eukaryotic viruses: retroviruses such as HIV, undergo a Latent (non-lytic) cycle in which the viral DNA (provirus) becomes inserted into the host’s DNA

Viral Mutations

DNA Viruses

• use host proofreading machinery

• error rate is very low ~ 1/10^8-11 bases

• herpsviruses genome range from 1.3 × 10^5 to 2.0 × 10^5

RNA viruses

• noproofreading ability

• error rate is high ~ 1/10³-10^4

• HIV 1-2 mutations and SARS 3 mutations in every genome

Effects of Mutations

1. Many are lethal

2. increase antidrug resistance (drug resistant HIV)

3. changes in virulence

4. changes that allow the viruses to evade the hosts immune system

5. changes in host range

How to Prevent Viral Infection

Key to anti-viral drug development is that the drug must target a process essential for viral replication, and it must be active against the virus without being “toxic” to the host organism.

Bacteriophage

Viruses that infect bacteria

Bacteriophage Plaque Assay

• a quantative laboratory techinique used to enumerate and isolate infectious bacteriophages in a liquid sample

• a serial dilution of phages is mixed with host bacteria in molten top agar (overlay) and poured over a solid agar base

• as bacteria grow into a confluent lawn, the infected cells lyse and release phage progeny, which infects neighboring cells. this cycle creates clear, circular zones of cell destruction called plaques.

• measured in PFU/mL

Bacteriophage Infection Steps:

1. Absorption: receptors may be host pili, proteins, oligosaccharides, or lipopolysaccharides (LPS)

2. penetration: the bacteriophage genome penetrates through the tail tube into the host cell

3. translation and replication:

• early genes: repair the hole in the bacterial cell wall and block host proteinase and restriction enzymes

• replicate phage genome

• late genes: structural proteins

4. Assembly and release process: lytic infection or lysogenic (temperate) infection

bacteriophage structure and receptor attachment

core components;

• head (capsid): houses the viral genome (dsDNA)

• collar and core: central cylindrical sheath connecting the head to the base

• base plate: contains specialized spikes for firm anchorage

• tail fibers: flexible extensions responsible for initial host cell recognition

attachment sequence:

1. initial attachment: tail fibers recognize and bind to host outer membrane receptors

2. final attachment: spikes on the base plate attach securely to the bacterial cell envelope

3. penetration: the tail sheath contracts, driving the inner tube through the cell envelope and cell membrane to inject viral DNA into the cytoplasm

Assembly and Release Process: Lytic Infection

• after all bacteriophage “parts” have been produced, new bacteriophage particles are assembled.

• a copy of the phage DNA genome is packaged, a preassembled icosahedral head

• the tail and accessory structures are assembled, and a few molecules of lysozyme or a comparable enzyme are packaged into the tail base plate

• the remaining enzymes hydrolyze specific bonds in the murein or peptidoglycan layer of the cell wall and create pores in the inner membrane of the host, thus facilitating host lysis and bacteriophage release

• the host cell is killed by cell lysis. lysis of the host cell is tightly timed event.

Lysogenic (Temperate) Infections

lysogenic or temperate bactierophages infect their hosts, but instead of killing the host during a lytic infection their genome becomes integrated into a specific region of the host chromosome

after penetration, the viral DNA integrates into the host chromosome and replicates every time the cell copies its chromosomal DNA during cell division

Lysogenic cells divide, giving rise to lysogenic progeny. when the phage genome is integrated into a site in the bacterial chromosome, its called a prophage. (They can carry genes that alter the physical or functional characteristics of a host bacterium)

some bacteriophages encode transposase, allows the phage to insert into the chromosome randomly, others integrate into specific locations

during the prophage state, all of the bacteriophage genes are repressed except a gene encoding a repressor protein, which prevents the synthesis of enzymes and proteins required for the lytic cycle

can carry host genes from one bacterial cell to another in a process called transduction