RNA directed RNA synthesis
RNA directed RNA synthesis Lecture 6 Biology 3310/4310 Virology Spring 2018 Truth is ever to be found in the simplicity, and not in the multiplicity and confusion of things --SIR ISAAC NEWTON
Some RNA history • • • • • •
1935 - Stanley crystallizes TMV
• •
By 1959, RNA was identified in many animal viruses
1936 - TMV crystals contain 5% RNA 1944 - DNA is genetic material 1952 - Hershey-Chase experiment 1953 - Structure of DNA 1956 - TMV nucleic acid is infectious; first demonstration that RNA can be genetic material
1960s - studies on viral RNA replication begin
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Parvovirus
Hepatitis B virus VII
Retrovirus
Adenovirus Herpes simplex virus
Reovirus Rotavirus
Poliovirus
Influenza virus VSV Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Identification of RNA polymerases
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Identification of RNA polymerases
• Polymerase discovered in (-) strand virus particles
• Sequence alignments (GDD), synthesis of recombinant proteins • Crystal structures
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
ER 15
M2
5' c
Golgi
16
NEP/NS2
M1
3' MA CA NC PR
NS1
10b
11 MA CA NC PR RT IN
5' c
17
RNA in 18 the virus particle
12
• (-) strand RNA genomes: coated with protein
19
14
13
• (+) strand RNA genomes: naked (exceptions: retrovirus, coronavirus)
5' 5'
15 16
• dsRNA genomes
udios f Virology, 4e 16
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
NA
ER 15
5' c
9
M2
NEP/NS2
M1
3'
10b
Golgi
16
NS1
17 18
19
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Rules for viral RNA synthesis
• RNA genome must be copied end to end with no loss of nucleotide sequence
• Viral mRNAs must be produced that can be efficiently translated by cellular protein synthesis machinery
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Universal rules for RNA-directed RNA synthesis
• RNA synthesis initiates and terminates at specific sites on the template • RdRp may initiate synthesis de novo (like cellular DdRp) or require a primer
• Other viral and cell proteins may be required • RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in 5’-3’ direction
• Some non-templated synthesis Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Base DNA pol 5’3’-
Primer
P
OH
-3’OH
Template
-5’
Direction Of Chain Growth
Two-metal mechanism of polymerase catalysis
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus
Which is a universal rule about RNA directed RNA synthesis? A. B. C. D.
RdRp may initiate de novo or require a primer RNA synthesis initiates randomly on the RNA template RNA is synthesized in a 3’-5’ direction RNA synthesis is always template-directed
1
Sequence relationships among polymerases
•
Gly-Asp-Asp in (+) strand RNA polymerases
•
Asp-Asp in RT, segmented (-) strand polymerases
•
Gly-Asp-Asn in nonsegmented (-) strand polymerases
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
GDD
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Product
Template Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Structure of UTP bound to poliovirus RdRp
2’-OH
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
(+) strand RNA viruses
(Togaviridae - Sindbis, SFV, Chik)
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Poliovirus viral genome = mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
cis-acting RNA element (moveable)
Cellular polyadenylated RNAs not copied Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Vesicle formation in virus-infected cells
Uninfected HeLa cell
Flavivirus infected cell Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
PV-infected HeLa cell
Coronavirus-infected cell ©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus
Which is a part of the poliovirus replication strategy? A. B. C. D.
The production of subgenomic mRNAs De novo (without primer) initiation of RNA synthesis Circularization of template for initiation of RNA synthesis All of the above
2
(+) strand RNA viruses
(Togaviridae - Sindbis, SFV, Chik)
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Togaviridae viral genome = mRNA But not all of it is translated!
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press ©Principles of Virology, ASM Press
(-) Strand RNA viruses
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
VSV viral genome is not mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Unimolecular
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Influenza virus viral genome is not mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus How are influenza virus and VSV RNA synthesis similar? A. The switch from mRNA to genome RNA synthesis is controlled by an RNA binding protein B. Polyadenylation occurs at a short stretch of U residues C. Viral mRNAs are shorter than (-) genome RNA D. All of the above
3
dsRNA viruses Reoviridae: reovirus, rotavirus
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Reovirus (+) strand not accessible by ribosomes!
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Each dsRNA segment is attached to RdRp via the 5’-cap
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
6 7 5' c HA
NA
7
3'
ER
c
c
9
M2
NEP/NS2
M1
15 RNA directed RNA 3synthesis '
NS1
5' c
10b
Golgi
• • • • • •
(+) RNA, (-) RNA, dsRNA 16
18
Polymerase basics Site of RNA synthesis Genome replication
19
mRNA synthesis poly(A) addition
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
ScEYEnce Studios Principles of Virology, 4e
©Principles of Virology, ASM Press
Some RNA history • • • • • •
1935 - Stanley crystallizes TMV
• •
By 1959, RNA was identified in many animal viruses
1936 - TMV crystals contain 5% RNA 1944 - DNA is genetic material 1952 - Hershey-Chase experiment 1953 - Structure of DNA 1956 - TMV nucleic acid is infectious; first demonstration that RNA can be genetic material
1960s - studies on viral RNA replication begin
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Parvovirus
Hepatitis B virus VII
Retrovirus
Adenovirus Herpes simplex virus
Reovirus Rotavirus
Poliovirus
Influenza virus VSV Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Identification of RNA polymerases
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Identification of RNA polymerases
• Polymerase discovered in (-) strand virus particles
• Sequence alignments (GDD), synthesis of recombinant proteins • Crystal structures
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
ER 15
M2
5' c
Golgi
16
NEP/NS2
M1
3' MA CA NC PR
NS1
10b
11 MA CA NC PR RT IN
5' c
17
RNA in 18 the virus particle
12
• (-) strand RNA genomes: coated with protein
19
14
13
• (+) strand RNA genomes: naked (exceptions: retrovirus, coronavirus)
5' 5'
15 16
• dsRNA genomes
udios f Virology, 4e 16
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
NA
ER 15
5' c
9
M2
NEP/NS2
M1
3'
10b
Golgi
16
NS1
17 18
19
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Rules for viral RNA synthesis
• RNA genome must be copied end to end with no loss of nucleotide sequence
• Viral mRNAs must be produced that can be efficiently translated by cellular protein synthesis machinery
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Universal rules for RNA-directed RNA synthesis
• RNA synthesis initiates and terminates at specific sites on the template • RdRp may initiate synthesis de novo (like cellular DdRp) or require a primer
• Other viral and cell proteins may be required • RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in 5’-3’ direction
• Some non-templated synthesis Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Base DNA pol 5’3’-
Primer
P
OH
-3’OH
Template
-5’
Direction Of Chain Growth
Two-metal mechanism of polymerase catalysis
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus
Which is a universal rule about RNA directed RNA synthesis? A. B. C. D.
RdRp may initiate de novo or require a primer RNA synthesis initiates randomly on the RNA template RNA is synthesized in a 3’-5’ direction RNA synthesis is always template-directed
1
Sequence relationships among polymerases
•
Gly-Asp-Asp in (+) strand RNA polymerases
•
Asp-Asp in RT, segmented (-) strand polymerases
•
Gly-Asp-Asn in nonsegmented (-) strand polymerases
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
GDD
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Product
Template Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
Structure of UTP bound to poliovirus RdRp
2’-OH
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
(+) strand RNA viruses
(Togaviridae - Sindbis, SFV, Chik)
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Poliovirus viral genome = mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
cis-acting RNA element (moveable)
Cellular polyadenylated RNAs not copied Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Vesicle formation in virus-infected cells
Uninfected HeLa cell
Flavivirus infected cell Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
PV-infected HeLa cell
Coronavirus-infected cell ©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus
Which is a part of the poliovirus replication strategy? A. B. C. D.
The production of subgenomic mRNAs De novo (without primer) initiation of RNA synthesis Circularization of template for initiation of RNA synthesis All of the above
2
(+) strand RNA viruses
(Togaviridae - Sindbis, SFV, Chik)
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Togaviridae viral genome = mRNA But not all of it is translated!
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press ©Principles of Virology, ASM Press
(-) Strand RNA viruses
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
VSV viral genome is not mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Unimolecular
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Influenza virus viral genome is not mRNA
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Go to: b.socrative.com/login/student room number: virus How are influenza virus and VSV RNA synthesis similar? A. The switch from mRNA to genome RNA synthesis is controlled by an RNA binding protein B. Polyadenylation occurs at a short stretch of U residues C. Viral mRNAs are shorter than (-) genome RNA D. All of the above
3
dsRNA viruses Reoviridae: reovirus, rotavirus
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Reovirus (+) strand not accessible by ribosomes!
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
Each dsRNA segment is attached to RdRp via the 5’-cap
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
©Principles of Virology, ASM Press
6 7 5' c HA
NA
7
3'
ER
c
c
9
M2
NEP/NS2
M1
15 RNA directed RNA 3synthesis '
NS1
5' c
10b
Golgi
• • • • • •
(+) RNA, (-) RNA, dsRNA 16
18
Polymerase basics Site of RNA synthesis Genome replication
19
mRNA synthesis poly(A) addition
Virology Lectures 2018 • Prof. Vincent Racaniello • Columbia University
ScEYEnce Studios Principles of Virology, 4e
©Principles of Virology, ASM Press
