Replication of
ReverseTranscribing
Hepatitis B
Virus
Family Hepadnaviridae
• Hepatitis – inflammation of liver (injury,
chemical/drug, infectious agent)
• DNA virus – tropism for hepatocytes
• Unusual genome architecture and mode of
replication (DNA pol;reverse transcription)
• Difficult to grow in cell culture
• Found in:
– Humans (HBV)
– Animals (Woodchuck hepatitis virus)
– Plants (Cauliflower mosaic virus)
Genus: Hepadnavirus
• Mammalian, avian
• Hepatitis B virus (HBV) – “serum”
hepatitis, virus found and
transmitted in human blood & body
secretions, close contact
• Woodchuck hepatitis virus (WHV)
• Duck hepatitis B virus (DHBV)
HBV
• Envelope – HBS Ag (3 surface gp), 3545 nm
• Spherical inner core nucleocapsid –
HBC, HBE
• Incomplete viral forms (HBS Ag) found
in blood:
– spherical, fiber
– initially identified as “Australian Ag”
– 25 nm Dane particle
HBV Genome: dsDNA
• Small, circular, not
closed, partially ds
DNA
• Full-length (-)DNA
strand, 3.2 kb (5’ end
has covalently linked
viral protein)
• Partial (+)DNA
strand, 1.7 – 2.8 kb
(5’ end has covalently
linked viral RNA)
• Two 11 bp direct
repeat sequences
(DR1, DR2) required
for DNA replication
• Terminally redundant
ends (circularize)
HBV Genome: Four ORF
• Overlapping genes, no
splicing, 10 proteins
• ORF C – 2 core
proteins:
– HBC, HBE
• ORF P – 2 proteins
for DNA replication:
– DNA pol (RT/RNase)
– protein primer (-)DNA
synthesis
• ORF S – 3 surface
envelope gp:
– HBS (L, M, S)
• ORF X – 3 proteins:
– regulatory,
transactivation
HBV: Entry / Uncoating
• Receptor-mediated endocytosis
• Partial uncoating, viral DNA pol completes
dsDNA synthesis
• Viral dsDNA enters nucleus
• Ligate free ends, convert to covalently
closed circular dsDNA
• Associate with cell histones, similar to
episome DNA
HBV Genome Replication
• 3.4 kb mRNA transcript (larger than
genome), template for DNA genome
synthesis
• Viral protein primer for (-)DNA strand
synthesis by DNA pol (RT)
• Internal start site on mRNA
• As DNA synthesis occurs, RNA strand
degraded by RNase-H (RT)
• When DNA reaches end, “jumps” to
other end to complete synthesis
• Terminal repeat sequences important
for strand recognition
• Short viral RNA sequence primer for
(+)DNA strand synthesis by RT
HBV Assembly/Release
• Core particles form in nucleus
• Viral DNA genome inserted before
synthesis of (+)DNA finished
• Complete (-)DNA strand, with partial
(+)DNA strand
• Assembly with cell envelope in ER/Golgi
• Virus and small number of incomplete
membrane particles (HBS) released and
found in blood
HBV and Hepatocellular
Carcinoma (HCC)
• HBV infection increases risk (200x) for
HCC
• Following infection, HBV DNA persists as
episome or integrates into host cell DNA
• Modulated by viral X protein (transactivator)
• Chronic HBV infection with continued virus
replication, over time, results in liver
damage and cancer
• Host unable to clear virus by immune
defense (no appearance of HBS antibody)
Reading
• Chapter 21 – Hepadnaviruses:
Variations on the Retrovirus Theme
• Questions: 3, 4
Class Discussion –
Lecture 12
• 1. What is the evidence that HBV is a
retrovirus?
• 2. Why does hepadnavirus infection
sometimes lead to liver cancer?
Chapter 25: Viruses
and the Future –
Problems and
Promises
• “Imagination is more important
than knowledge.”
· Albert Einstein
PROBLEMS: Emerging Viruses
• HIV, Influenza, HCV – pandemics, high
mortality
• New, changing strains
• Breakdown of public health – war,
depression, poverty
• Social changes – legal, moral, truths
• It’s a small world – global economy,
international travel, interrelationships
• Environmental change – habitat, weather,
industry, pollution
• Are there solutions to these problems?
PROBLEMS:
Bioterrorism and Fear
• Classes of Bioterrorism Agents
– Class A – Ebola Virus, Smallpox virus
– Class B – Encephalitis Viruses
– Class C – Sin Nombre Virus
• What is our best protection?
• “I am not dreaming of Utopia, only of a
world in which problems are not resolved
by force but by intelligence, good will and
equity;
• a world in which killing, no matter the
reason, and the destruction of a fellow
man’s life or home, is a crime, a world in
which our youth will not have to spend
their best years studying organized
manslaughter,
• in which neither force nor megatons nor
poison gases will decide a nation’s standing
but the sum of its knowledge, its ethics,
the gifts it makes to mankind, the
happiness it gives to men, the measure in
which it lifts human life.”
-Albert Szent-Gyorgyi
“Do not be overcome by evil,
but overcome evil with good.”
• Paul the Apostle (c.5 – c.67)
PROMISES:
“Trojan Horse” Virus
• Use VSV (good) to overcome HIV (evil)
• Recombinant VSV:
– Remove VSV envelope gene
– Insert cell CD4, CXCR4 genes (cell receptors
for HIV); express as envelope proteins on VSV
• Novel VSV will attach to HIV gp120
• In cell culture, novel VSV infects and kills
HIV-infected cells
PROMISES:
Phage Factor
• Phages produce lysins (enzymes) to exit
bacteria
• Use lysins to treat bacteria infection (strep
throat, endocarditis, meningitis, pneumonia)
• 2012 Clinical Trials: CF-301 lysin against
MRSA (methicillin-resitant Staphylococcus
aureus)
• Scientific American.com/aug2012/phage
PROMISES:
Going Viral
• Use M13 bacteriophage to generate
electricity
• Piezoelectricity: mechanical energy into
electrical energy
• Biomolecules such as protein and nucleic
acids are piezoelectric when compressed
• Press down on virus film attached to
electrodes to produce electricity
• Apply to cell phone, pacemaker, etc.
• Safe, cheap, easy to create
Why Study Virology?
• Viruses important members of our
biosphere
• Biology “writ small”
– Principles learned
– Apply to all life
“The future is a world
limited by ourselves.”
• Maurice Maeterlinck (1882-1949)
– Nobel Laureate in Literature
MICR 401 Final Exam
•
•
•
•
•
Tuesday, Dec. 4, 2012
1:30 – 3:00pm
Papovavirus thru Hepadnavirus
Case Study #9-15
Lecture & Discussion Questions, Reading &
Chapter Questions
• Exam:
– Objective Questions (MC, T/F, ID)
– Short Essay Questions
QUESTIONS???
ALL I REALLY NEED
TO KNOW
ALL I REALLY NEED TO
KNOW I LEARNED IN
KINDERGARTEN
• Robert Fulghum
ALL I REALLY
NEED TO KNOW
• about how to live and what to do and
how to be I learned in kindergarten.
• Wisdom was not at the top of the
graduate-school mountain, but there
in the sandpile at Sunday School.
These are the things I
learned:
•
•
•
•
Share everything.
Play fair.
Don’t hit people.
Put things back where you found
them.
• Clean up your own mess.
• Don’t take things that aren’t yours.
• Say you’re sorry when you hurt
somebody.
• Wash your hands before you eat.
• Flush.
• Warm cookies and cold milk are good
for you.
• Live a balanced life – learn some and
think some and draw and paint and
sing and dance and play and work
every day some.
• Take a nap every
afternoon.
• When you go out
into the world,
watch out for
traffic, hold
hands, and stick
together.
• Be aware of wonder.
• Remember the little seed in
the Styrofoam cup:
• The roots go down and the
plant goes up and nobody
really knows how or why,
but we are all like that.
• Goldfish and hamsters and white
mice and even the little seed in the
Styrofoam cup – they all die.
• So do we.
• And then remember the Dick-andJane books and the first word you
learned – the biggest word of all –
•
LOOK.
LOOK
• Fulghum, Robert.
• 1988. All I Really Need To Know I Learned
In Kindergarten: Uncommon Thoughts On
Common Things. New York: Villard Books.
• 2003. 15th Anniversary Edition. All I Really
Need To Know I Learned In Kindergarten:
Reconsidered, Revised & Expanded, With
Twenty-Five New Essays. New York:
Ballantine Books.
ALOHA
Kauai, Hawaii