BIOL 2421 Microbiology

BIOL 2421 Microbiology Lecture Notes: Viruses Chapter 11 Dr. Weis

Viruses: are not considered living organisms as they are acellular and are inactive outside the host. Sometimes thought of an obligate intracellular parasite, they also can be viewed as inert biochemical complex of organized macromolecules. They are nonmotile and are entirely dependant on physical factors for chance movement and spread to infect other susceptible cells.

A complete virus is called a virion and consists of a nucleic acid enclosed in a protein coat. Virus and virion are used interchangeably in most books.

Viruses can exist in two phases:

a) Extracellular: cannot reproduce independently, few enzymes present

b) Intracellular: replicates nucleic acids, induces host metabolism for enzymes to synthesize virion components.

Therefore, their simple structural organization is characterized by:

1) contain single nucleic acid (DNA or RNA) called the viral genome

2) protein coat that surrounds nucleic acid (NA)

3) Multiply inside cells by using substances: ATP, enzymes

4) Synthesize structures that can transfer viral NA to other cells

Cultivation for Identification

* chick embryo

* tissue culture: animal cells or bacteria or plant cells

Purification for ID and study

* centrifuge: differential, gradient

* precipitation

* denature

* assay: hemagglutination, plaque, PCR

Viral Host Range

Spectrum of host cells a virus can infect (broad or narrow)

Most infect specific types of cells on one host species

Requirements:

~ attach at specific host receptor sites

bacteria = cell wall, fimbria, flagella

animal = plasma membrane receptors

~ certain host cellular factors are needed for viral replication

proteins, enzymes, free NA, tRNA, ribosomes, ATP

Size: smaller than bacteria, range from 20-1000 nm length and 10-400nm diameter. Most must be viewed with scanning /transmission electron microscopes.

Structure

Parts of the Virion

^ NA : DNA or RNA : contain genes for capsid and key genes for initiating its lifecycle

ss (single strand) or ds (double strand)

linear or circular

can be in segments

the combination of viral NA can be as follows

ds DNA: linear or circular, majority of DNA virus are double stranded

ss DNA: linear or closed circle

ds RNA: segmented

ss RNA: majority of RNA viruses are single stranded, many are segmented

if strand same as mRNA = (positive) + strand

if strand is complimentary to mRNA = (negative) – strand

^ Capsid

Tight protein coat that surrounds NA

Protein coat is coded by viral NA

Identical symmetrical structural building units of the protein coat are called protomers

May contain enzymes necessary for NA replication

Capsid Shapes:

Icosahedral= regular polyhedron with 20 equilateral triangles

Helical = hollow tube like cylinders, rigid or flexible

Complex = other structures such as a tail or multiple layers

* Bacteriophage: sheath, base plate, pin, tail fiber

* Poxviruses

Can also have capsomeres, ringed protein protomer subunits arranged in groups (5 or 6 subunits) around the outside of the capsid that forms as an extension of the capsid. Capsomere proteins can be the same or different proteins than the capsid. Capsomeres aid in :

protecting from harsh environmental conditions : pH , temp

enzymes for cell penetration

immune response

NA + capsid = nucleocapsid or genocapsid

^ Envelope

lipid + protein + CH20

covers capsid in some viruses, forms a spherical shape

sometimes forms as a leftover host cell plasma membrane that

had been modified to contain a mixture of normal and viral proteins/glycoproteins coded via NA from virus or host

+ /- spikes (peplomers)

* CH20 + protein projections from envelope

* help to ID viruses

* used to help viruses attach

* may possess enzymes to help in penetration

If envelope is lost, so is virulence/ infectivity

NOTE: If no envelope, called non-enveloped virus (or naked) viruses. Sometimes naked is used when there is no protein coat surrounding the nucleic acid, as seen in plant viruses. To avoid confusion, use the word "non-enveloped".

Viral Taxonomy:
Host Range: Animal, Plant, Bacteria

NA characteristics, Capsid symmetry, Nucleocapsid diameter

+/- envelope, number of capsomeres

location of viral replication, immunologic prosperities, special features

Disease caused, method of transmission

Bacteriophages

Viruses that infect certain strains of bacteria

Most have ds DNA

Classified as to morphology

a) tailless icosahedral

b) Contractile tails

c) Noncontractile tails

d) Filamentous

Lifecycle: Lysogenic, Lytic

Lytic Cycle: seen with virulent bacteriophages, lyse their host cells

* landing and attachment via tail fibers to specific receptor sites

* tail contraction, penetration, unplugging, DNA injection (ATP)

lysozyme enzymes released to breakdown cell wall

capsid and other structures remain outside as a “ghost”

* Biosynthesis

host DNA/RNA/protein synthesis stops, host DNA is degraded by viral enzymes coded for by viral proteins

early mRNA made to direct synthesis of enzymes for viral DNA replication, uses old host nucleotides

late mRNA directs enzyme synthesis for structural proteins, assembly, and cell lysis.
3 capsid proteins are made for construction of tail, tail fibers, heads.

* Maturation: virus is spontaneously assembled

(assembly line style)

nucleocapsid made first, then baseplate/tube/sheath

added. Finally a collar (at neck) and tail fibers

* Release: lysis of bacteria

lysozyme made via viral genome attacks host cell wall peptidoglycan

another protein attacks bacterial plasma membrane

osmosis causes swelling and eventual rupture

approximate time for lytic cycle (burst time) : 20-40 minutes

Bacterial defenses:

1) Bacterial mutations can change receptor sites used by phages for recognition

2) Bacterial restriction enzymes can recognize and cut up foreign DNA

RNA bacteriophages

ssRNA codes for enzymes to create RNA replicase

creates temporary dsRNA, each acting as a template

several RNA copies made

+RNA for viral genome

mRNA for translation of viral proteins

Lysogenic Cycle: seen with temperate bacteriophages, co-exist

Virus is latent until it initiates a lytic cycle

* attach to cell wall

* penetration

* integration -> virus now called a prophage inserted at specific site

viral genome does not take control of host

does not destroy host while producing new phages

viral genome replicated along side of host DNA

creates clones of infected cells that appear normal

can change phenotype of host that is not directly

related to life cycle of virus

may or may not be an actual part of host DNA, if

not part of DNA, usually initiates a lytic cycle

* induction:

lytic cycle starts when viral genome excised

triggered when host is unable to survive

3 results of lysogeny

a) surviving hosts are immune to reinfection

b) phage lysogenic conversion -> host acquires new properties

c) specialized transduction: bacterial genes are picked up during

excision of viral genes and transferred to another bacterium

Viruses of Eukaryotes: Animals

Classification: Virus morphology, chemical nature, disease caused, location, genetic relationships, individuals who discovered

One group: based on system affected second classification based on viral families.

Viral Multiplication/Replication in Animals

1) Attachment of virus reactive protein sites to host plasma membranes at glycoproteins. Passive reaction and specific for binding.

Damage (chemical) or blocking (antibody neutralization) of the viral binding sites can render virions noninfectious

Host receptors are genetically determined, may be found in all or local / specific cells

host receptors are necessary for function/role:

maybe site for hormone binding or other molecules

that involve the immune system signaling/reception

complementary receptors on host to viral attachment sites

viral attachment sites vary:

a) icosahedron fibers on capsid

b) envelope spikes

c) bottom of a surface or depression of viral capsid

2) Penetration

a) direct penetration: seen with nonenveloped viruses

binds to receptor

change in capsid structure allows the NA to enter the cytoplasm

b) fusion: seen with special enveloped viruses that have certain protein fusion glycoproteins

fusion with plasma membrane to create a pore

nucleocapsid taken into cytoplasm

c) receptor mediated endocytosis: seen with enveloped viruses

attach to coated pits

forms coated vesicles

fuses with lysosomes of host that begin uncoating

3) Uncoating

varies depending on virus

separation of nucleocapsid :

removal of capsid

release of viral NA

accomplished by one of three ways

a) lysosome enzymes inside vesicle

b) viral coded enzymes

c) via cytoplasmic enzymes of host

4a) Biosynthesis of DNA Viruses

early genes take over host cell to synthesize DNA, RNA

may inhibit host DNA or may stimulate macromolecule synthesis

normally, viral DNA replication occurs in the host cell nucleus

during the S phase of the normal host cell cycle

early mRNA is transcribe from viral DNA via host enzymes

ds (+/-) DNA à DNA – strand directly transcribed to viral mRNA

ss (+) DNA à converted to ds DNA and the (-) DNA used

ss (-) DNA à converted to ds DNA and the (-) DNA used

General Summary for DNA virus transcription/translation

Viral DNA is replicated via viral enzymes in host nucleus, usually

If viral DNA is inserted in the host genome called a provirus.

Host enzymes are used for synthesis of capsid and other proteins

Early genes-> proteins for DNA synthesis (RNA and DNA polymerase)

Late genes -> proteins for capsid synthesized by free ribosomes

Assembly of nucleocapsid occurs in nucleus, usually

Proteins + viral DNA = virions

Virions move to endoplasmic reticulum for release

If enveloped, glycoproteins synthesized by the RER are sent to the Golgi

Golgi vesicles transport glycoproteins to PM to form clusters at viral exits

Some viruses do not use the host PM for their envelope, but rather the nuclear membrane (seen in Herpes viruses)

4b) Biosynthesis of RNA Viruses

similar to DNA viruses: attach, penetrate, uncoat

RNA replication usually in cytoplasm

Only (+) mRNA strand can be used to synthesize proteins

more diverse that DNA in strategies for replication and translation

Sometimes put into classes (Class III, IV, V, VI) to create the 4 RNA virus groups:

Class IV ss (+) RNA à synthesis from positive sense strand is analogous to mRNA, used to make proteins use viral replicase to make ss (-) RNA as template for more ss (+) RNA

Class V ss (-) RNA à transcribe +sense from antisense (-) strand for use as mRNA. Uses (-) RNA via viral replicase to make +RNA , then makes copies of more ss (-)RNA.

Class III ds (+/-) RNA à one strand sense (+) other antisense (-). Uses transcriptase to make mRNA à proteins mRNA used to make (+ /-) RNA copies via replicase enzymes.

Class VI 2 ss (+) RNA à has own enzymes:

RNA dependenta DNA polymersase and reverse transcriptase

ss (+) RNA+ à (-) DNA complimentary à ds (+/-) DNA à mRNA and ss (+) RNA

5) Viral Maturation (assembly)

late genes code for capsid proteins

capsid assembly usually in cytoplasm, can be in nucleus

procapsid forms, nucleic acid inserted after condensing

viral components held together by weak bonds (hydrogen)

accumulation of virion assembly may form inclusions which are visible (microscopically)

6) Viral Release

Naked virion released by host cell lysis

Enveloped virion released by budding

virion pushes through host plasma membrane.

Other internal membrane sources can be the nucleus, E.R. and Golgi

Does not necessarily destroy the cell during viral exit

Host Cell Damage causing cytocidal (cell death) results or degenerative changes causing cytopathic effects, which eventually end in cell death

1) Inhibit host cell DNA, RNA, and protein synthesis – cell death

2) Damage host cell lysosomes resulting in release of hydrolytic enzymes – cell death

3) Alter plasma membrane via insertion of viral proteins – immune system destroys

4) Increases in viral protein concentrations have toxic effect – cell damage/death

5) Inclusion bodies formed, disrupt cell structure – cell death

6) Chromosomal changes / disruptions – cell death

7) Host cell transformed into a malignant cell, if immune system attack – cell death

Viral Infections

Acute: rapid onset, last for a short time

Chronic: persistent, lasting many years, replicate at slow rate over a few years

Detectible, eventually fatal. Many are related to brain degeneration.

(Measles, Rubella, Papovirus, HIV, Echovirus)

Latent: stops reproducing, dormant, no symptoms until reactivated. (Herpes)

At equilibrium and not reactivated until stress or decr immune response

Slow: extremely slow developing, replication lasting many years

Integration into host genome may be reason for chronic or latent viral infections

If virus integrated into animal genome, called a provirus and sets up a lysogenic state where the new cells (via mitosis) carry viral properties and are considered infected as well or may create cancerous cells.

Diagnosis of Viral Infections

A) Direct: detection and demonstration of the virus itself

* growth amplification in culture

* electron microscopy

* serological techniques

1) precipitation

2) agglutination

3) immunofluorescence

4) ELISA

5) Compliment fixation

6) Radio immuno assays

7) purified vial antigens

* molecular techniques

1) Gel electrophoresis

2) Western blotting

3) PCR

4) Southern blotting

5) Genome sequencing

B) Indirect: study of host’s response to that virus

(see previous host response to damage)

Emerging Viruses

Existing viruses that have expanded their host territory

Arise due to:

1) Changes to cause disease in individuals only immune to viral ancestor

2) Spreads from one host species to another (monkeypox à humans)

3) Disseminates from small area to more widespread (hantavirus, AIDS)

4) Increased exposure: isolated areas exposed à roads, deforestation

See textbook and other charts for comparison of

Bacteriophage to Eukaryotic phage, DNA viruses, RNA viruses

Viruses and Cancer

Cancer = uncontrolled cell growth

a) Proto-oncogenes promote growth

b) Tumor-suppressor genes control proto-oncogenes

Changes in either or both can lead to uncontrolled cell growth

Neoplasia = “new form” caused by abnormal cell growth

Benign if localized, remains in one place

Malignant if actively spreads causing secondary tumors

Anaplasia = “without form” causing a reversion back to a primitive state

Cancer Causes:

Diet

Carcinogens (mutagens)

Genetic (oncogenes related to cell growth factors)

Viruses: can alter genetic material and turn “on” oncogenes (initiation) as

a result of other changes [genetic, chemical, physical]

or can inactivate a tumor suppressor

Viral DNA integrates into host DNA and replicated ~ lysogeny

Provirus never excised

10% of cancers are known to be virally induced

Transformation : acquire properties distinct from normal

create antigens on surface: TSTA on PM; T antigen on nucleus

change cells à shape abnormalities; chromosome abnormalities

DNA oncogenic viruses

* Adenovirus

* Herpes

Epstein-Barr Virus à 2 cancers: carcinoma, lymphoma of the jaw

* Pox

*Papo

Papillomas à cervical cancers

*Hepadenoviruses

Hepatitis B à liver cancer

RNA oncogenic viruses

Retroviruses à 2 types of adult HTLV leukemias

Virus Inhibition

by the immune response

* Phagocytosis

* Antibody neutralization

* Lymphocytes (T-cells)

* viral vaccines (inactivated, attenuated)

by treatment with antiviral drugs

* prevent attachment

* prevent uncoating

* act as a nitrogen base analogue for substitution in the genome

* inhibit reverse transcriptase

* cell protection by inhibiting viral replication via interferons

by physical or chemical agents

* heat (moist and dry)

* UV light

* chlorine

* iodine

* heavy metals

* formaldehyde / gluteraldehyde

* lipid soluble detergents

Prions

Proteinaceous infectious particle

Neurological Disease = spongioform encephalopathy (holes in the brain)

Loss of coordinated movements

Progressive degeneration, eventual death

Cause conversion of normal host glycoprotein (PRPC) to cellular prion protein (PRPSC). Still searching for a related/hidden NA to code for conversion or some very small virus hidden within these proteins.

Will affect cell adhesion, recognition, and uptake, primarily in neurons in the brain

Brain neurons have the PRPC protein which protects them from free radical damage.

PRPC on membrane reacts with PRPSC which converts normal to abnormal as it folds into a different form consisting of clumps of pleated sheets.

These abnormal proteins are taken in and accumulate in lysosomes creating plaque as the lysosomal enzymes are unable to degrade the changed protein. Empty areas of dead tissue begin to appear in the brain causing clinical signs.

Prion based neurological diseases

* Scrapie (sheep and goats)

* BSE (mad cow disease)

* CJD (Creutzfeldt-Jakob disease)

* GSS (Gerstamnn-Sträusler-Scheinker Syndrome)

* kuru

* fatal familial insomnia

Plant Viruses

Similar to animal viruses

Based on morphology

Most are RNA viruses

Rod shape capsid with spiral capsomeres

Horizontal or vertical transmission

Horizontal

Plant receives virus from an external source

Many require insect vectors as plants are protected by cell walls

Viruses can also enter through injured epidermal sites on the plant

Vertical

Inherits viral infection from parent

Asexual propagation

Sexual reproduction via seeds

Once infected, spreads from cell to cell via plasmodesmata

Cause: color change, deformed or stunted growth, wilting

Viroids

Circular, ssRNA (naked as they are not covered by a protein coat)

Self splicing, self replicating.

Does not code for any proteins (act like an intron in eukaryotic genome)

Disrupt normal plant metabolism, development, growth by causing

errors in regulatory systems that control gene expression

Transmitted between plants

Fungi and Algae Viruses

ds RNA

mycoviruses induce disease symptoms and eventual host destruction and lysis

Insect Viruses

ds DNA or ds RNA

Inclusion bodies formed in host either in nucleus or cytoplasm

Can be latent infections