Transcript General Virology

General Virology
VIRUS STRUCTURE
Virion vs virus
• Virion is the infectious
particle
– composed of nucleic
acid, protein capsid,
+/- envelope
– may be extracellular or
intracellular
• Virus is any stage of
infection
Capsid
• Functions
– Protection of NA
– Attachment for naked
viruses
– Enzyme
• Helical vs Icosahedral
Symmetry
• Tobacco mosaic virus is a
ssRNA virus composed of
6000 nucleotides. The
capsid is made of 2100
copies of a single protein
subunit that contain 158
amino acids.
Icosahedral symmetry
• 20 identical equilateral
triangles
• Structural units on faces
to give morphological
capsomers
Envelope
• Attachment
• Entry
• Assembly- matrix
proteins
• Release
• Proteins are viral
• Lipids are host
• If the membrane envelope is
destroyed, the virus
becomes noninfectious.
Why?
Genome - DNA or RNA
• Sense:
Positive-sense, Negative-sense,
Ambi-sense
Number:
single, segmented
• strandedness - (single)
(double)
• linear or circular,
partial double stranded
circle
Proteins
• structural proteins
• non-structural virion
proteins
– transcriptase,
– protease
– integrase
WHAT IS A VIRUS?
Viruses :
acellular organisms
Genomes obligately replicate inside host
cells using host metabolic machinery to
different extents, to form a pool of
components which assemble into particles
called virions.
F A virus differs from a cell in three fundamental ways:
i
A virus usually has only a single type of nucleic acid
serving as its genetic material. This can be single or double
stranded DNA or RNA;
ii Viruses contain no enzymes for energy metabolism, thus
cannot make ATP;
iii Viruses do not encode sufficient enzymatic machinery to
synthesize their component macromolecules, specifically, no
protein synthesis machinery.
ONE STEP GROWTH CURVE
•
•
•
•
•
1939- Ellis and Delbruck:
Infection with a high multiplicity
of infection (MOI): ratio of virus
to host cell
– Simultaneous infection
– Single replication cycle
Sample at time intervals by plaque
count for plaque-forming units
(PFU),
Identification of latent phase
Determination of burst size/viral
yield
Detection
F Viruses cannot
be grown on sterile
media, but require
the presence of
specific host cells.
Virology and Human Disease
Tuesday, August 12
Viruses
• Infectious agents found in virtually all life forms
including humans, animals, plants, insects, and bacteria
• Virion Structure
– genetic material (either DNA or RNA, double or single
stranded, linear or circular)
– Protein coat (capsid) surrounding genetic material
• Rod (helical), polyheldral, or more complex
– with or without a lipid envelope
• Viruses are not free living
– Unable to reproduce themselves outside of a living cell
– Transmit their genetic information from one cell to another
• Viruses often damage or kill the cells that they infect
Figure 18.1 Size of a virus, a bacterium, and a eukaryotic cell
Figure 18.2 Viral structure
Rod-shaped
Icosohedral
Enveloped
Complex
Figure 18.3 A simplified viral reproductive cycle
• Obligate intracellular
parasites
– Rely on the equipment of
cell to replicate
– Host range = viruses can
only infect certain cell
types
• Dependant on recognition of
host cell receptor
• Entry, uncoating,
replication, release, cell
lysis, infection of new
cells
Bacterial Viruses
• Bacteriophages – phages
– Virulent phage
• Reproduces only by a lytic life cycle
– Lytic – results in the lysis of the host cell
» Phage T4 (infects E. coli)
– Temperate phage
• Reproduce by either lytic or lysogenic life cycle
– Lysogenic - replication does not destroy host
» Phage λ (infects E. coli)
Figure 18.4 The lytic cycle of phage T4
Figure 18.02x2 Phages
Figure 18.5 The lysogenic and lytic reproductive cycles of phage , a
temperate phage
Animal Viruses
• Virus Classification
– Genome
•
•
•
•
DNA or RNA (+/- sense)
Size (kb)
Single or double stranded, linear or circular
# segments, sequence
– Morphology
• Virion size and shape
• Plus or minus envelope
• Capsid symmetry and structure
– Protein
– Biological properties
– Physical and chemical properties
Figure 18.6 The reproductive cycle of an enveloped virus
• Glycoproteins on envelope
recognize receptor on host
cell
• Viral envelope fuses with
cell membrane
• Genome and capsid enter
the cell
• Genome is copied
– New RNA genomes
– mRNA translated into capsid
proteins and glycoproteins
for envelope
• Capsid assembly
• Virus buds from cell
Viral Entry: receptors and fusion
• Initial attachment – binding to host cell
– Viral surface protein recognizes receptor
• Carbohydrates
• Lipids
• Proteins – transmembrane
• Entry – virion conformational change in response
to receptor or pH
– Location of entry
• Plasma membrane (neutral pH)
• Endosomal membrane (acidic pH)
– Type of entry
• Fusion – enveloped viruses
• Penetration – nonenveloped viruses
Modification of Host Cell Function
• Effects on cellular translation
– Viruses activate PKR (cellular kinase) which suppresses
cellular translation
• Effects through receptor binding
– Viral receptor binding may mimic the effects of the natural
ligand
• Induction of cell proliferation
– Viruses need replication machinery and induce cells to
enter the cell cycle upon infection
• Effects on cellular RNA processing
– Inhibit cellular transcription
– Degrade cellular mRNAs
– Alter RNA processing or export
Viral Replication
• DNA viruses
– Replicated and transcribed similar to host cell
– Examples = adenovirus, herpes virus
• RNA viruses
– Plus stranded
• Translated directly (therefore RNA is infectious)
• Virally encoded RNA-dependant RNA polymerase (RdRp)
synthesizes more genomic RNA and mRNA for proteins
• Example = poliovirus
– Minus stranded
• Must be transcribed by RdRp for replication and transcription of
viral mRNA
• Enzyme is carried by the virus into the cell during infection
• Example = influenza, measles
• Retroviruses – HIV
Figure 18.02x1 Adenovirus
Virus infects the
upper respiratory
tract (common
Figure 18.x6 Herpes
• 8 herpesviruses infect humans
• Human diseases
–
–
–
–
–
Oropharyngeal and genital lesions – herpes simplex
Chickenpox – varicella zoster virus
Congenital microcephaly – CMV (growth retardation)
Burkitt’s lymphoma – EBV (childhood tumor) (mononucleosis)
Kaposi’s sarcoma – KSHV
Viral Replication
• DNA viruses
– Replicated and transcribed similar to host cell
– Examples = adenovirus, herpes virus
• RNA viruses
– Plus stranded
• Translated directly (therefore RNA is infectious)
• Virally encoded RNA-dependant RNA polymerase (RdRp)
synthesizes more genomic RNA and mRNA for proteins
• Example = poliovirus
– Minus stranded
• Must be transcribed by RdRp for replication and transcription of
viral mRNA
• Enzyme is carried by the virus into the cell during infection
• Example = influenza, measles
• Retroviruses – HIV
Figure 18.x3 Paralytic Polio
• Summer time epidemic disease – summer of 1885 in England
• More than 20,000 cases each year in the U.S.
• Infection spreads to brain and CNS and replicates in muscle cells, spreading to
motor neurons and causing paralysis
• Poliovirus was cultured – Robbins, Enders, and Weller received the Nobel Prize
in 1954
• First vaccine developed in 1955 by Salk was inactivated virus
• Live attenuated vaccine (Sabin) was approved in 1961 and eliminated virus from
the Americas
Viral Replication
• DNA viruses
– Replicated and transcribed similar to host cell
– Examples = adenovirus, herpes virus
• RNA viruses
– Plus stranded
• Translated directly (therefore RNA is infectious)
• Virally encoded RNA-dependant RNA polymerase (RdRp)
synthesizes more genomic RNA and mRNA for proteins
• Example = poliovirus
– Minus stranded
• Must be transcribed by RdRp for replication and transcription of
viral mRNA
• Enzyme is carried by the virus into the cell during infection
• Example = influenza, measles
• Retroviruses – HIV
Simpler infectious agents - Prions
• Infectious proteins – causing degenerative brain diseases
– Scrapie in sheep
– Mad cow disease in cows
– Creutzfeldt-Jakob disease in humans
• Prions are misfolded forms of normal proteins in the brain
• Prions induce normal proteins to convert to prion form
triggering a chain reaction that increases their numbers
Figure 18.10 A hypothesis to explain how prions propagate
What are viruses?
•
Small obligate intracellular parasites
•
Virion
– Complete virus particle : nucleic acid + protein coat, which may be surrounded by
an envelope
– It is the form in which the virus moves between cells or hosts
•
Viral Genome
– EITHER RNA or DNA genome surrounded by a protective virus-coded protein
coat (Capsid)
•
Propagation depends on specialized host cells supplying the machinery for
replication, metabolism and biosynthesis
• The DNA or RNA genome may be :
– ss – single stranded or
– ds – double stranded
• Genomes may be either:
– (+) sense: Positive-sense viral RNA is identical to viral mRNA and thus
can be immediately translated into protein by the host cell.
OR
– (-) sense: Negative-sense viral RNA is complementary to mRNA and thus
must be converted to positive-sense RNA by an RNA polymerase before
translation.
Definitions
• Bacteriophage
– Virus that infects prokaryotic (bacterial) cells.
• Nucleocapsid:
– viral nucleic acid + the protein coat that encloses it.
– Represents the packaged form of the viral genome.
Viral Structure - Overview
Nucleic acid
Capsid
Nucleocapsid
Envelope protein
Viral
Membrane
envelope**
Spike
protein
protein
Viral Structure
• Varies in size, shape and symmetry
• VIP for classification
• 3 types of capsid symmetry:
– Cubic
(icosahedral)
• Has 20 faces, each an equilateral triangle. Eg. adenovirus
– Helical
• Protein binds around DNA/RNA in a helical fashion eg.
Coronavirus
– Complex
• Is neither cubic nor helical eg. poxvirus
http://micro.magnet.fsu.edu/cells/virus.html
Viral Structure
Figure 1 An array of viruses. (a) The helical virus of rabies. (b) The segmented helical virus
of influenza. (c) A bacteriophage with an icosahedral head and helical tail. (d) An enveloped
icosahedral herpes simplex virus. (e) The unenveloped polio virus. (f) The icosahedral HIV
with spikes on its envelope.
Viral Replication
• When a virus infects a cell, nucleic acid must be uncoated and gain access
to metabolic machinery of cell.
• Virus life cycle is characterized by:
– attachment
– penetration, with entry of nucleic acid into cell
– early expression of virus genes (either directly by translation, if virus contains
"+" RNA, or indirectly after transcription and then translation)
– replication of virus nucleic acid
– synthesis of new virion components
– packaging and assembly of new virions
– exit from cell
• Attachment
– specific binding of a virion protein (the anti-receptor) to a
constituent of the cell surface (the receptor)
• e.g. hemagglutinin of influenza virus
• some complex viruses (HSV) may have more than one
species of anti-receptor molecule
• Penetration
– energy-dependent step
– occurs almost instantaneously after attachment
• After the virus attaches to the host cell, it can enter the cell
by several mechanisms:
– Transfer of the entire viral particle across the cell
membrane by endocytosis
– Transfer of only the viral genome through the cell
membrane
– Fusion of the viral envelope with the host cell membrane
•
Uncoating
– at same time as penetration or shortly after
– separation of viral nucleic acid (n.a.) from outer structural components
• Released as:
– free n.a. (picornaviruses)
– as nucleocapsid (reoviruses) = may need acidic pH in endosome
– viruses only infectious agent for which dissolution of infecting agent obligatory step
in replicative pathway
•
Expression of viral genome and synthesis of viral components
• After the viral nucleic acid is released inside the host cell:
– The transcription and translation processes of the host cell are
redirected for the production of viral proteins and nucleic acids
– The different types of nucleic acid genomes are expressed and
replicated in several ways:
• DNA genomes undergo replication-using processes similar to
cellular replication
• RNA genomes may be +ssRNA; Can be read directly as an mRNA
or reverse transcribed by reverse transcriptase into DNA
• RNA genomes may also be -ssRNA; The RNA must first be used
as a template to form +mRNAs
Assembly and Release
• Components of capsid synthesis directed by late genes
• Assembly of enveloped viruses needs interaction with plasma
membrane which has been modified
• Final stage of infection
• Enveloped viruses released gradually by budding or exocytosis
• Naked viruses accumulate in cytoplasm and released during lysis
Prions
• Prions
–
–
–
–
–
Infectious particles that are entirely protein.
No nucleic acid
Highly heat resistant
Animal disease that affects nervous tissue
Affects nervous tissue and results in
• Bovine spongiform encepahltits (BSE) “mad cow disease”,
• scrapie in sheep
• kuru & Creutzfeld-Jakob Disease (CJD) in humans
•
Vaccination
Edward Jenner developed the first vaccine in 1798 for smallpox
Edward Jenner
Microorganisms as the Medical Enemy
• the other side of the picture
– influenza: 1918-1919
• killed 20 million people
Micrograph of Influenza virus
Medical Microbiology Successes
• smallpox
– last known disease in the world was documented in 1977
– believed at one time prior to eradication, that 80% of the
world’s population would be affected by smallpox
Smallpox virus
Clinical Manifestation of Smallp
Prions
• prions
– contain only protein
– causative agent for some neurodegenerative
diseases in humans and animals
Prion
What is a Virus?
• Definition: “Poison, venomous secretion”
• Obligate intracellular parasites
– Dependent on other “HOST” organisms
– Produced from assembly of pre-formed components
rather than “growing” or division
• Viruses are composed of nucleic acid (DNA or
RNA) surrounded by a protective protein coat
(Capsid)
• There are viruses that can infect every type of
living cells (animal, plant, bacteria, etc.)
• Are they ALIVE??
History of Virology
• 1796 - Edward Jenner vaccinated a boy, James
Phipps, with material from a cowpox lesion on the
hand of a milkmaid. The boy became immune to
small pox!
• 1885 - Louis Pasteur experiments with rabies
vaccination. He originates the terms "virus" and
"vaccination" and develops the scientific basis for
Jenner's experimental approach to vaccination
Louis Pasteur
(1822-1895)
History of Virology
• 1892 –Dmitri Iwanowski shows that extracts
from diseased tobacco plants can transmit disease
to other plants after passage through filters fine
enough to retain the smallest known bacteria.
– This is generally recognized as the
beginning of Virology! But, nobody
understood the significance until…
Dmitri Iwanowski
(1864-1920)
• 1898 – Beijernick made the same discovery,
but suggested that the pathogen is a distinct
agent, not just really small bacteria
Martinus Beijerinick
(1851-1931)
History of Virology
• 1911 - Francis Peyton Rous demonstrated that a
virus (Rous sarcoma virus) can cause cancer in
chickens. Rous is the first person to show that a
virus could cause cancer in animals.
• 1935 - Wendell Stanley crystallizes tobacco
mosaic virus and shows that it remains
infectious.
Polio
Francis Peyton Rous
(1879-1970)
TMV
History of Virology
• 1937- The first to propagate yellow
fever virus in chick embryos and
successfully produced an attenuated
vaccine.
• 1940 - Helmuth Ruska used an electron
microscope to take the first pictures of
virus particles.
• 1949 - John Enders, Thomas Weller and
Frederick Robbins were able to grow
poliovirus in vitro using human tissue
culture.
Max Theiler
(1899-1972)
History of Virology
• 1955 -The Salk vaccine against polio is
introduced into general use.
Jonah Salk
• 1979 - The W.H.O. officially declared
smallpox to be completely eradicated!
– First microbial disease ever to be completely
eliminated
Smallpox EM
History of Virology
• 1981 – First recorded cases of AIDS,
mostly in homosexual populations
• 1983 - the discovery of human
immunodeficiency virus (HIV), the
causative agent of AIDS.
Virus Classification
• A universal system for classifying viruses, and a
unified taxonomy, has been established by the
International Committee on Taxonomy of
Viruses (ICTV) since 1966. The system makes
use of a series of ranked taxons:
• Family (-viridae) (eg. Retroviridae)
– Subfamily (-virinae) (e.g. Orthoretrovirinae)
• Genus (-virus) (e.g. Lentivirus)
• Currently there are 30,000 - 40,000 known viruses
• BUT – there are 106 virus particles per milliliter of
seawater – most are unknown!
Virus Size
•
Most viruses vary from
30-200nm in length
Viral Composition
• All virions (extracellular virus) are composed of:
– Nucleic acid genome core - the genetic material!
– Capsid composed of viral proteins to protect genome
Viral Capsids
• Capsids are composed of repeating units of Viral proteins,
and serve to protect the viral genome
• Generally, capsids are formed by very regular, symmetric
patterns
• Most capsids are either HELICAL or ICOSAHEDRAL
Capsid structure: Helical
• Repeating
structure of
proteins
surrounding viral
genome
Example:
Tobacco Mosaic Virus
(TMV)
Viral Genome
(DNA or RNA)
Capsid
Proteins
Capsid Structure: Icosahedral
• An Icosahedron is a 20sided structure
• Viral genome at the
core
Example:
Adenovirus
Capsid Proteins
(Genome in the
center)
Viral Structure: Envelope
Some Viruses also contain
• Envelope:
– Lipid bilayer (membrane) derived from host
membranes, surrounding capsid
– Contains Viral Glycoprotein “spikes”
• Matrix:
– Protein layer between Capsid and Envelope
Viral Structure: Envelope
Matrix
Examples:
Influenza virus
HSV (herpes simplex)
Glycoproteins
Capsid
The Basics
• Entry – The virus must find/enter into a cell
• Making copies of virus parts
– Replication of viral genome
– Gene expression of viral genes
• Exit
– Assembly of viral components
– Leave cell; go on to infect new cells
Entry - Steps
• Attachment
– Tethering the virus to the cell
• Penetration
– Crossing the plasma membrane
• Uncoating
– Releasing the genome
Entry - Attachment
• (Glyco)proteins on the surface of the virus bind to
receptor proteins on cell surface
• “Tethers” the virus to cell surface
Entry - Penetration
• Crossing the PM
• Several different strategies:
– TRANSLOCATION of the entire virion across the cell
membrane; eg. Poliovirus
– ENDOCYTOSIS of the virus into intracellular
vacuoles; eventually into the cytoplasm. Eg.
Adenovirus (unenveloped), Influenza virus (enveloped)
– FUSION of the viral envelope with the cell membrane.
Requires the presence of a viral fusion protein in the
virus envelope; eg. Retroviruses, Herpesviruses
Entry - Uncoating
• Release of the GENOME by (partial) removal of capsid
proteins surrounding genome
• Depending on site of replication, genome may be released
into cytoplasm or translocated into the nucleus
Entry – Different Strategies
http://www.mssm.edu/micro/vintro3.gif
Replication/Gene Expression
After the genome has entered the host cell, two simultaneous
steps occur:
• Replication of the genome
• Transcription and translation of viral genes (gene
expression)
How?
Why are these steps necessary?
DNA Replication - Overview
http://library.thinkquest.org/04apr/00217/images/content/74-Summary-DNA-Replication.jpg
Gene Expression - Overview
http://stemcells.nih.gov/StaticResources/info/scireport/images/figurea6.jpg
RNA processing/splicing
- Overview
• Transcribed RNA is modified to make mature mRNA:
5’ cap
3’ poly-A tail
Replication/Gene Expression
Two things to consider in replication (dependent on type of viral
genome)
• Location
– Nucleus
– cytplasm
• Type of enzymes used for replication and/or gene expression
–
–
–
–
DNA polymerase
DNA-dependent RNA Polymerase
RNA-dependent RNA Polymerase
Reverse transcriptase (RNA-dependent DNA polymerase)
SOME VIRUSES NEED TO BRING THE
NECESSARY ENZYMES WITH THEM!
Gene Expression
• Gene expression is tightly regulated in viruses in order to
control viral replication
• Viruses need to use cell machinery (i.e. ribosomes, tRNAs,
etc.) to synthesize proteins
• Viral mRNAs contain “normal” cellular control signals
– ribosome-binding sites
– splice signals
– polyadenylation signals
Gene Expression
• Usually, viruses first express low levels of regulatory
proteins (enzymes, etc.)
• Later in infection, viruses express tons and tons of
structural proteins – i.e. proteins that get included in virus
(e.g. capsid protein, glycoproteins, etc.)
Gene expression
• Most viral proteins get made/assembled in cytoplasm
• Envelope proteins get made into ER and “travel”
through endomembrane system
- Glycoprotein
Assembly
• Involves the assembly of all the components
necessary for the formation of the mature virion
• Assembly may be spontaneous, or driven with the
help of cellular/viral proteins that are not components
of the mature virions
• The site of assembly varies for different viruses:
– Picornaviruses, Poxviruses, Reoviruses - In the cytoplasm.
– Adenoviruses, Herpesviruses, Parvoviruses - In the
nucleus.
– Retroviruses - On the inner surface of the cell membrane.
Egress
Non-enveloped Viruses:
• Cell lysis - releasing the newly made virions in the
extracellular matrix
Enveloped Viruses:
• Budding – acquiring a viral envelope
– This process is generally coupled with egress, eg.
Capsid budding into the PM
Egress - Budding
HIV:
Putting it all together
Typical lifecycle of Unenveloped
virus:
Putting it all together
Typical lifecycle of
Enveloped virus:
Lytic vs. Lysogenic
Many Transcription Strategies
Don’t worry about virus names (in red).
Introduction to Virology
• The cultivation of viruses is complex and includes three
common methods
– Chicken egg culture
– Cell culture
– Animal inoculation
ATTACHMENT
Click after each step to view process
PENETRATION
UNCOATING
HOST
FUNCTIONS
Transcription
Translation
REPLICATION
VIRAL
LIFE
CYCLE
ASSEMBLY
(MATURATION)
RELEASE
93
MULTIPLICATION
1.
Protomers
2.
Capsomers
4.
MatureC
apsid
3.
ProCapsid
PRION Protein
Normal Folding Pattern
of PrP
Alternate Folding
Pattern of PrPSc