Biol 3400

Tortora et al - Chap 13

Virology

What is a virus?

How do viruses differ from cellular microorganisms?

  1. Acellular
  2. consist of a particle consisting of a nucleic acid surrounded by a protein coat.
  3. In extracellular form a virus particle or virion is metabolically inert
  4. A variety of sizes and shapes – 20 nm (Poliovirus is 28 nm in diameter or the size of a ribosome) to 1000 nm (Smallpox virus is about 200 nm in diameter; Ebola virus can be up to 1000nm in length)
  1. Viral Genomes can be used to classify viruses
  2. viral genome - RNA or DNA; ss or ds
  3. linear (T4, T7) or circular dsDNA (SV40)
  4. linearssDNA (X174)
  5. linearssRNA (Q) or dsRNA (6)
  6. May have one or more nucleic acid molecules (segmented – Reoviruses - 10 dsRNA molecules or influenze virus – 8 ssRNA molecules)
  7. One group uses both RNA and DNA but at different stages of their life cycle
  8. This variety of form of the hereditary molecule is unique to viruses suggests that different viruses evolved from different host cells possibly as genetic extensions
  9. Genomes often contain unusual nitrogenous bases – T-even phages of E. coli contain 5-hydroxymethylcytosine
  10. Size ranges from approximately 4 kb (Q) to 670 kbp (bacteriophage G)
  1. Protein coat or capsid
  2. Single or several types of protein - protein subunits (protomers) that are formed into larger assemblies or structural subunits called capsomers. Information necessary for proper folding and aggregation is contained within the structure of the proteins so that they are capable of self-assembly
  3. nucleocapsid = capsid and genome
  4. Helical (rods) or isometric symmetry (spherical or icosahedral – 20 triangular faces and 12 corners), binal = helical and isometric (e.g., T-even bacteriophage) or complex (pox viruses)
  5. The capsid morphology is an important taxonomic criterion
  6. Viral particle may also contain one or more virus-specific enzymes (e.g., lysozyme, RNA-dependent RNA polymerase, reverse transcriptase, neuraminidases) that are important in the infection and replication processes
  7. Enveloped viruses - some viruses are further surrounded by a membrane envelope
  8. membrane (phospholipids, proteins and carbohydrates) layer surrounding the capsid
  9. membrane does not function like the membrane of a living organism
  10. proteins are typically virus-specific and coded by the virus genome. May be important in attachment of the virion or release of the virion from the host cell
  11. may function in initial attachment or protection of host immune system
  12. acquisition of envelope is part of maturation process - acquired after assembly of nucleocapsid - e.g., upon leaving cell
  1. Require a host to replicate - independent activity
  2. Viruses can be classified according to hosts (i.e., animal, plant, bacteria,…)
  3. Host range is generally very specific

I.Viral Taxonomy

  • Much less satisfactory state than that of either bacteria or eukaryotic microorganisms
  • International Committee for Taxonomy of Viruses – 1971 – 2000 virus species placed in 3 orders, 73 families, 9 subfamilies and 287 genera.
  • Definition of families - greatest weight placed upon

i) nucleic acid type

ii) nucleic acid strandedness

iii) the sense of ssRNA genomes

iv) presence or absence of an envelope

v) symmetry of capsid

vi) dimensions of the virion and capside

orders end in virales; family names end in viridae, subfamily, in virinae; and genus and

species in virus

e.g., poxviruses – family Poxviridae; subfamily Chorodopoxvirinae (poxviruses of vertebrates); Genus Orthopoxvirus – variola major (smallpox), vaccinia and cowpox

Detailed List of Taxonomic characteristics

1 . Nature of host

2. Nucleic acid characteristics

  • DNA or RNA (never both)
  • ss or ds
  • molecular weight
  • segmentation and number of pieces of nucleic acid (RNA viruses)
  • sense of the strand in ssRNA

3. Capsid symmetry – helical, isometric, binal

4. Presence of envelope and ether sensitivity

5. Diameter of virion

6. Number of capsomers in isometric viruses

7. Immunological properties

8. Gene number and genomic map

9. Intracellular location of replication

10. The presence or absence of a DNA intermediate (ssRNA viruses) and the presence of reverse transcriptase

11. Type of virus release

12. Disease caused and/or special clinical features – method of transmission

II.Viral Cultivation and Purification

  • Require host cells for cultivation – inoculate suitable hosts or use tissue culture
  • Some plant viruses are difficult to work with because they require the whole plant

Viruses are

  • large relative to proteins
  • more stable than normal cell components
  • have surface proteins.

These properties allow the use of techniques designed for isolation of proteins and organelles including

  • Differential and density gradient centrifugation
  • Precipitation of viruses (e.g., ammonium sulfate, polyethylene glycol)
  • Denaturation of contaminants (e.g., chloroform)
  • Enzymatic digestion of cellular constituents

Viral Assays

How do you quantify the number of virions in a suspension?

i) Direct count with an electron microscope (virus containing sample is mixed with known concentration of small latex beads and sprayed on a coated specimen grid) – not easy

ii) Indirect count

a)Hemagglutination assay – many viruses can bind to the surface of red blood cells – if the ratio of virions to RBC is large enough then they agglutinate. The hemagglutination titre is the highest dilution of virus that still causes hemagglutination

b) Quantify number of infectious units by measuring effects on hosts

  • A virus infectious unit is the smallest unit that causes a detectable effect when added to a susceptible host – as few as one virion
  • The number of infectious units per volume is referred to as the titre
  • Plaque assay – when a virus initiates infection on a layer or lawn of host cells growing out on a flat surface, a zone of lysis or zone of growth inhibition may occur that results in a clear area (plaque) in the lawn of growing cells. It is assumed that each plaque originated from replication events that began with one virion. Viral titres are expressed as plaque-forming units (PFU).
  • Plating efficiency - counts made by plaque assays are always lower than counts made with electron microscope. Plating efficiency with bacteriophage is usually > 50% but with some animal viruses may be <1%
  • The plaque procedure may be used to prepare pure viral strains
  • Cell cultures may also be used to titre virus – infection with viruses leads to cellular deterioration - known as the cytopathic effect

iii) Whole animal methods – some viruses do not cause recognizable effects in cell culture and yet kill animals.

  • Quantification in these systems relies on preparing serial dilutions of unknown samples and injecting these into several sensitive animals.
  • After a suitable incubation period, the number of live and dead animals at each dilution is determined and an end point dilution is calculated. This may be the dilution that kills 50% of the animals – Lethal Dose killing 50% of the animals = LD50

III.Viral Multiplication

A.Requirements

  1. Host cell (a cell within which a virus replicates) must be permissive and virus compatible with host cell

Host range

  • Permissive or compatible host cells permit viral replication
  • Nonpermissive host cells do not permit viral replication
  • Host range may be broad (a number of species) or narrow (one cell type of a single species)
  • e.g., bacteria, archaea, fungi, protozoa, plants and animals are infected by viruses
  1. Host cell does not degrade virus
  2. Viral genome contains necessary information for modifying host metabolism
  3. Virus must be able to use the host metabolic activities to produce new virus particles
  • Viral genome achieves control of cell’s metabolic activities – viral protein synthesis dominates over host cell protein synthesis

How? - Host transcription is inhibited or

Viral mRNA’s are translated more efficiently than host cell proteins

  • Virus uses host’s ribosomes, ATP and reduced coenzymes for viral progeny (virion) production

B.Outcomes of infection

  1. Abortive infections – host cell is nonpermissive or viral progeny are incapable of infecting other host cells
  2. Restrictive infections – host cells are transiently permissive – virus persists in cell until it becomes permissive or only a few cells in a population produces viral progeny at any time. Viral genome may persist within a host cell without destroying it
  3. Productive infections – viral replication occurs within permissive cells – produces virions

C.Stages of viral Multiplication

  • General strategy conserved for most viruses
  • In the first few minutes of infection the virus genome is separated from the capsid and the virion no longer exists as an infectious entity. This is referred to an eclipse (Fig 13.10).
  • Maturation begins when newly synthesized genomes are packaged into protein coats – during this phase the titre in cells increases
  • Latent period includes the eclipse and maturation periods – newly synthesized virions are not detected outside the cell
  • At the end of maturation release of mature virions occurs
  • The number of virions released for a particular virus and cell type is call the burst size and can vary from several to several thousand.
  • The replication cycle varies in length from 20 to 60 minutes for bacteriophage to 8 to 40 h for animal viruses
  1. Adsorption - Attachment to host cells – energy independent process that requires sufficient ion concentration. Binding sites or receptors are typically glycoproteins but may also be proteins, carbohydrates, lipids, lipoproteins or complexes of these. Viruses attach only at specific receptor sites – host range or specificity

Lambda – Maltose binding protein

HIV – CD4 protein on T-helper cells and macrophages

Poliovirus – neuronal cellular adhesion molecule

Rabies virus – acetylcholine receptor on neurons

  1. Penetration - entry into host cells – depends upon type of virus
  • transport of entire virus across the cytoplasmic membrane – endocytosis
  • simultaneous penetrating and uncoatingmay occur for some viruses (e.g., T-even bacteriophage) resulting in only the viral genome entering the cell
  • fusion of viral envelope with host cytoplasmic membrane
  • Rather elaborate mechanisms may be used by complex viruses – e.g., T4 bacteriophage attaches to a bacterial cell with the aid of tail fibres. The fibres retract and brings the tail pins in contact with the cell surface. Lysozyme like enzymes produce a small hole in the peptidoglycan cell wall. The tail sheath contracts and the viral DNA passes into the cytoplasm though a hole in the tip of the phage tail.

Uncoating - Release of viral genome from the capsid

  1. Biosynthesis of viral proteins and viral genome – essential for viral replication

viral proteins - alter host function, replication of viral genome, viral capsid proteins, and packaging proteins

DNA virusesreplication - theta and rolling circle

multiple initiation points for linear genomes

semiconservative replication

  • dsDNA () viruses – transcription of minus strand – host polymerases or viral encoded factors
  • ssDNA (+) viruses – replicate other DNA strand to produce dsDNA intermediate - transcription of minus strand – host polymerases or viral encoded factors

RNA viruses

i)Terminology – mRNA is said to be in the plus (+) configuration and the complement of mRNA is in the minus (-) configuration

ii)Cellular RNA polymerases are DNA dependent (i.e., use DNA as a template), therefore RNA viruses require an RNA-dependent RNA polymerase

  • ssRNA (+) viruses – genome can serve as mRNA encodes for synthesis of virus-specific, RNA-dependent RNA polymerase as well as the template for synthesis of (-) strand that is used for replication of (+) strand

e.g., picornaviruses such as polioviruses

  • ssRNA (+) Retroviruses – produce DNA by reverse transcription during replication. Genome is two identical single-stranded (+) RNA molecules. Capsid carries RNA genome and reverse transcriptase. Linear dsDNA enters nucleus and integrates into the chromosome as a provirus (that never leaves the host chromosome).

e.g., HIV

  • ssRNA (-) viruses – transcription using (-) strand template to form (+) strand mRNA. Carry virus-specific, RNA-dependent RNA polymerase in virion that uses + strand as a template to produce viral RNA.

e.g., Rhbdoviruses

  • dsRNA () viruses – Reoviruses – transcription using (-) strand template to form (+) strand mRNA – carry virus-specific, RNA-dependent RNA polymerase in virion.

4. Assembly (Maturation) of virions

  • New viral particles are assembled in host cell from newly synthesized components
  • Packaging – nucleic acid genome is placed into capsid nucleocapsid
  • Assembly occurs in a variety of locations (specific to type of virus)  cytoplasm and nucleus of cells
  • Further modifications for enveloped viruses – e.g., addition of envelope and viral proteins to phospholipid envelope (e.g., influenza virus, herpesvirus)

5. Release of viruses from host cell (lysis)

  • Many virus particles are produced within a cell
  • Release often kills host cell
  • Release may involve lysis with the aid of viral encoded enzymes – e.g., lysozyme
  • Exit may be achieved through budding exocytosis or fusion of vesicles containing virus particles with cytoplasmic membrane

IV.Variations in Viral Multiplication

A. Lambda ()

  • Bacteriophage are classified as virulent (lytic) and temperate. Virulent phage infect host cells and undergo the lytic cycle thereby killing their host cells. In contrast, temperate phage can cause the lytic pathway or enter a state of lysogeny, where the phage genome is integrated into the host genome. The phage genes are not expressed and the phage genome (prophage) is replicated along with the host chromosome.
  • A phage-encoded repressor protein maintains the state of lysogeny. This repressor protein controls expression of lytic genes on the prophage as well as prevents any incoming phage genes from being expressed thereby imparting immunity to infection by the same type of virus.
  • Review the lytic and lysogenic pathways (Figure 13.12)
  • is a temperate phage that infects Escherichia coli. It is a dsDNA phage with a genome size of 48.5 kb. The genome is linear except for a single stranded 12-nucleotide tail found on the 5’ end of each strand. These single stranded regions are complementary and in the host cell anneal and the DNA is ligated to form a double stranded circle. attaches to cell surface receptor (LamB). Immediately upon entry into the cell the  genome circularizes and expression of phage genome begins. Cells that contain a prophage are generally immune to reinfection by the same type of phage (but not other types of phage). Lysogeny may also results in the host cell exhibiting new properties. This is known as phage conversion. Examples of phage conversion include cells expressing toxins associated with disease such as the diphtheria toxin (Corynebacteriumdiphtheriae) and Shiga toxin (Escherichia coli O157:H7).

Control of LyticvsLysogenic pathways

  • Two regulatory proteins control which pathway occurs in 
  • If Cro is made in high amounts then lambda is irreversibly committed to the lytic pathway. If the lambda repressor (cI) dominates then lysogeny will occur.
  • Agents that damage DNA (UV radiation) as well as certain chemicals (e.g., antibiotics such as mitomycin C) induce lysogens to produce phage (i.e., undergo the lytic pathway). One of the E. coli cell’s response to DNA damage is to produce the RecA protein, which destroys the lambda repressor thereby allowing phage specific transcription to begin

Review Generalized and specialized transduction (Chapter 8)

B. Animal viruses

  • The basic process of viral multiplication is the same.
  • Attachment (adsorption)
  • protein and glycoprotein receptors of the plasma membrane. Receptors are inherited characteristics of host.
  • Viral attachment sites are generally distributed over the surface of the virion.
  • Entry
  • Viruses enter by pinocytosis. Enveloped viruses may enter by fusion in which the viral envelope fuses with the host cell membrane and releases the capsid into the cell’s cytoplasm
  • Uncoating
  • Enzymatic removal of the viral capsid – enzymes may be host or virus in origin depending on virus species
  • Biosynthesis
  • This process varies depending on the virus (DNA vs RNA)
  • Generally DNA containing animal viruses replicate DNA in the host cell nucleus using viral enzymes and the capsid and other proteins are synthesized in the cytoplasm using host cell enzymes. The proteins migrate to the nucleus for assembly with DNA. Virions are transported along the endoplasmic reticulum to host membrane for release
  • RNA viruses – differences in biosynthesis due to how mRNA and viral RNA are produced – refer to RNA virusessection above on page 6.
  • Maturation and release
  • In enveloped viruses, the envelope develops around the virion by a process called budding. Envelope proteins, encoded by the viral genome, are incorporated into host cell plasma membrane. The host cell may not die immediately as a result of the budding process
  • Nonenveloped viruses are released through rupture in the host cell membrane – usually resulting in the death of the host cell

C.Plant viruses and viroids

  • Morphologically similar to animal viruses – some can multiply in insect cells
  • The cause of diseases in many economically important crops
  • Plant cell wall serves as a protective barrier against viruses – viruses must enter through wounds or be transmitted by insect, nematode or fungal vectors
  • Some plant diseases are caused by viroids – short pieces of naked RNA only 300 to 400 nt long and with no associated protein coat (e.g. potato spindle tuber viroid). The RNA does not code for any proteins. Current research has discovered similarities between viroid and intron nucleotide sequences.

V.Prions

  • Protein infectious particle or Prion(Protein infectious only) is a term coined by Stanley Pruiner. Prions are misfolded proteins believed to cause a small number of related infectious diseases known as Transmissible Spongiform encephalopathies (TSE).
  • TSE are a group of diseases of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. All of the diseases are also characterized by a long incubation period without symptoms and the lack of a measurable immune response. These include bovine spongiform encephalopathy (a.k.a. – mad cow disease) and Creutzfeld-Jakob disease (CJD - a variant that affects humans). A new variant of CJD or vCJD is believed to result from humans eating infected tissue from cattle with BSE.
  • The diseases have neuropathological features that appear to be caused by accumulation of misfolded or an abnormal isoform of the normal cellular protein (prion protein or PrP, also referred to PrPc for PrP cellular). The misfolded form is referred to as PrP – scrapie (PrPsc) or PrP-res (due to partial resistance to protease K). Vacuolation of the CNS often accompanies PrPsc accumulation in the final stages.
  • The brain is heavily affected while the rest of the body is relatively unharmed. Neurons in infected tissues develop large vacuoles giving the brain tissue a sponge-like appearance.
  • A prion is an infectious proteins (devoid of informational nucleic acids) that is capable of converting a normal host protein PrPc into a prion protein.
  • The disease is hypothesized to be caused by a variant form of a naturally occurring glycoprotein (prion protein - cellular or PrPc). When PrPc is converted into the infectious form PrPsc (prion protein – scrapie; scrapie is a TSE that affects sheep) it adopts a very different three dimensional structure (misfolded structure) that has been suggested to promote the misfolding of PrPc resulting in prion protein

The “Protein-only” hypothesis