Transcript Virus Replication Cycle - Cal State LA

Human Viral Disease;
Virus Replication Cycle
Human-Virus Interaction
• Virus extinction
• Clear virus, immunity
• Large number of deaths
• Small Population
– Favors persistent virus infection
– Virus infection with an
immunological naïve person
– i.e. herpes simplex virus, parent
to newborn
• Large Population
– Many susceptible to infection
– Virus infected individuals
available all the time
– Sporadic spread of virus
– i.e. common “cold” virus, school
class room
Patterns of Virus Disease
• Asymptomatic infection – no disease
symptoms
• Acute infection – disease symptoms
• Persistent infection – long term
– Chronic: infectious virus
– Latent: no virus replication, virus reactivation
• Transformation – alter cell regulation,
tumor production, cancer
– No infectious virus
– Viral DNA, complete or partial
DNA Virus Infections
RNA Virus Infections
Acute Infection: Varicellazooster virus (VZV)
• Herpesvirus – one virus,
two diseases
• Varicella virus
• Chickenpox; common
childhood disease
• Primary acute
mucosal/skin infection
• Resolves in 1-2 weeks
• Virus infect and latent in
nerve cells
Persistent Chronic Infection:
VZV
•
•
•
•
Zooster virus
Shingles
Latent in nerve tissue
Presence of viral
DNA, no infectious
virus
• Virus held in check by
host immune defense
• Later in life,
reactivation of virus,
replicates, descends
down nerve tissue,
replicates in skin cells
One-Step Virus Replication
In Cell Culture
• High level of virus infection (1-10
virus/cell)
• Synchronous virus replication in cells
• All events required for cell infection
Virus Replication
Cycle
• Attachment (Adsorption)
• Entry / Uncoating
(Penetration)
• Gene Expression
(Synthesis: Early,
Genome, Late)
• Assembly (Maturation)
• Release (Lysis, Budding)
Virus Attachment
(Adsorption)
• Contact and interaction of virus to
host cell
• Recognition of virus to host cell
• Virus molecule that binds to host cell
called ligand (viral protein or
glycoprotein)
Attachment: Host Cell
• Virus binds to host cell
molecule - receptor
(i.e. cell protein,
glycoprotein, lipid)
• Receptors are
molecules that have a
role in normal
functioning of the cell
Attachment: Cell
Receptor
• Virus may bind up to three different
cell receptors in succession:
– Low affinity receptor - in high
abundance, virus contacts cell surface
– Primary receptor - in lower
concentration
– Co-receptor – follows binding of
primary receptor
Attachment: Specificity
• Host Range - the organism(s) that
the virus is able to infect (narrow or
wide) i.e. plant, animal, human
• Tissue Tropism- the cell type(s) a
virus is able to infect i.e. skin, oral,
GI, CNS
Attachment: Binding
• 3-D fit between viral ligand and cell
receptor
• Mainly weak electrostatic charges.
• Evidence for this is interaction may
require:
– specific pH
– specific ionic strength
– presence of specific ions i.e. Ca++, Mg++
Attachment: Nonenveloped
Picornavirus
• Virus ligand - a deep cleft (“canyon”) in
triangular face of capsid (viral proteins
VP1, VP2, VP3)
• Binds to cell receptor ICAM –1
(intracellular adhesion molecule 1), normal
function is to bind cells i.e. WBC
Attachment: Nonenveloped
Virus to Host Cell
Membrane
Attachment: Enveloped
HIV Virus
• Host cell protein in virus envelope
(cyclophilin A) initially binds HIV to
low affinity receptor (heparin
sulfate) of the cell
• Followed by binding of viral ligand
(gp120) to primary receptor (CD4)
on T helper cells, macrophages, and
glial cells
• Binding of gp120 to CD4 results in
conformational change of gp120,
which then binds to chemokine coreceptor CXCR4 on T lymphocytes
or CCR5 on macrophages
Attachment: Enveloped Virus
to the Host Cell Membrane
Entry / Uncoating
• Entry is the mechanism used by the
virus to penetrate into the host cell
• Uncoating is the separation of the
nucleic acid from the capsid, and
refers to changes that occur to make
the viral nucleic acid ready for
expression
Entry: Nonenveloped Virus
• Receptor-mediated
endocytosis
• Clathrin coated pits
(seen by EM)
• Invagination, pinch
off membrane
• Forms intracellular
endosome, contains
the virus
• Endosome becomes
acidified
Uncoating: Nonenveloped Virus
• Acid pH causes
conformational
changes in capsid
protein
• Hydrophobic region
interacts with
membrane, forms a
pore
• Viral nucleic acid
released
Entry / Uncoating:
Nonenvelpoed Poliovirus
Nonenveloped Virus:
Endocytosis
Entry / Uncoating:
Enveloped Virus
• Receptor mediated
fusion of virus
envelope with cell
plasma membrane
• Two modes of entry:
– Direct entry (pH
independent)
– Receptor-mediated
endocytosis (pH
dependent; for
uncoating)
Direct Entry / Uncoating:
Enveloped Sendai Virus
• At cell surface
by a viral
fusion protein
(active upon
cleavage)
• Viral capsid
released into
cytoplasm
Fusion at the Cell
Membrane: Enveloped
Virus
Entry By ReceptorMediated Endocytosis:
Enveloped Influenza Virus
• Lower pH in endosome
• Conformational change in
HA of influenza exposes
a fusion peptide
• Fusion of viral envelope
with endosomal envelope
• Release capsid into
cytoplasm
Influenza Virus Envelope
: Cell Membrane
Endocytosis: Enveloped
Virus
Receptor-Mediated
Endocytosis: Enveloped
Virus
Synthesis: “Early” Gene
Expression
• Release of viral genome into cell
(cytoplasm or nucleus)
• Virus regulates host cell metabolic
machinery
• Only some viral genes expressed (“early”
transcription & translation)
• Viral regulatory proteins and enzymes for
initial synthetic events
Synthesis: Genome
Replication
• Replication of viral nucleic acid
• Cellular or viral polymerase
• New genome synthesis “signals” for
additional viral synthetic events
Synthesis: “Late” Gene
Expression
•
•
•
•
Further expression of viral genome
“late” transcription and translation
Some regulatory proteins
Mainly structural (capsid, envelope)
proteins for progeny virus
Assembly (Maturation)
• This phase of viral replication is
FUNDAMENTALLY DIFFERENT from
organisms
• Viruses assembled from component parts,
not from division of a pre-existing virus
i.e. not exponential growth kinetics, but
“burst” of new virions
Self Assembly
• Concentration of viral structural
proteins and genomes (“reactants”)
adequate
• Self forming process (recognition
between viral components)
• Assembly follows basic laws of
thermodynamics
Virion Assembly
• Assembly requires protein-protein
interactions and protein-nucleic acid
interactions
• The order of assembly occurs two
ways:
– The genome serves as a focus for
assembly of the capsid surrounding it
(helical viruses)
– A hollow capsid formed and then filled
with the genome (icosahedral virus)
Assembly – Helical Virus:
TMV
• Rigid helical virus
• Composed of RNA plus identical
capsomers arranged in a helix
• TMV capsid proteins only recognize
TMV RNA
• This means that the protein-nucleic
acid interactions are very specific
TMV Assembly: Proteins
• First, 34 capsid
proteins assemble into a
pair of disks
• The outer portions
interact to hold the two
disks together, while
the inner portion has a
gap where RNA binds
• When the RNA enters,
the gap is closed to hold
the RNA in place
TMV Assembly: Genome
• RNA interacts with
the disks beginning
at the “pac”
(packaging signal)
site, which is about
1000 bases from the
3’ end of the genome
• The “pac” site
consists of ~ 500
bases that can form
a series of hairpin
loops
Summary: TMV
Assembly
•
•
•
•
•
Capsomers
Disc
Multiple helical disc
RNA binds to disc
Helix elongation of RNA
through central hole
Assembly: Icosahedral
Virus
• Has 20 triangular faces and each
face is composed of 3 subunits (or
multiples of 3). The subunits may be
identical or different
Assembly: Poliovirus
• Protomer is made with
Vp0, VP1, and VP3
• Five protomers combine to
form a pentamer
• Twelve pentamers combine
to form an empty
procapsid (60 protomers)
• RNA enters the procapsid
• A maturation cleavage
converts VP0 into VP2 and
VP4 to form intact virion
Cell Lysis
• Virus lytic infections cause distinct
changes of infected cell
• Changes called cytopathic effect (CPE) and
include:
–
–
–
–
–
Inclusion body
Nuclear pyknosis (shrinking)
Vacuole
Apoptosis
Syncytia (multinucleated cells)
Inclusion (Negri) Body Rabies Virus
Syncytia (“giant” cell)
Formation - Herpesvirus
Virus Release: Cell Lysis
• CPE usually secondary result of changes in
host cell metabolism by viral replication
• Virus may halt or alter host cell DNA
synthesis, transcription, and/or protein
synthesis (translation)
• Results in disintegration of infected cell
and release of progeny virus
Virus Release: Budding
(Exocytosis)
• Synthesis and insertion of viral
glycoproteins in host cell membrane
(nuclear, ER, Golgi, plasma membrane)
• Assembly of viral nucleocapsid
• Nucleocapsid and virus modified membrane
brought together (capsid protein may
interact directly with viral glycoprotein or
via a viral matrix protein)
• Exocytosis, or budding - may or may not
kill the cell
Virus Budding Through
Cell Plasma Membrane
Polarized Cell Plasma
Membrane Exit
• Viral envelope proteins
contain apical or basolateral
plasma membrane transport
signals
• Virus that bud apically tend
to cause localized
infections (release via
surface)
• Virus that bud basolaterally
tend to cause systemic
infections (release via
interior)
Reading & Questions
• Chapter 4: Patterns of Some Viral
Diseases of Humans
• Chapter 6: The Beginning and End of
the Virus Replication Cycle (omit
Questions 3, 4)
QUESTIONS???
Class Discussion – Lecture 3
• 1. How does an acute virus infection
differ from a persistent (chronic, latent)
infection?
• 2. Is virus attachment/entry similar to a
normal cell process?
• 3. How is the capsid of a helical virus
(TMV) assembled?
• 4. How is the capsid of a spherical virus
(poliovirus) assembled?
• 5. Non-enveloped virus are able to selfassemble in vitro, but not enveloped
viruses. Why?