RNA viruses: genome
replication and mRNA
production BSCI 437
Lecture 12
• Mechanisms of viral RNA synthesis
• Switch from mRNA to genomic RNA
production
General comments
All RNA viral genomes must be efficiently
copied to provide
• Genomes for assembly into progeny virions
• mRNAs for synthesis of viral proteins.
Two essential requirements common to RNA
virus infectious cycles:
1. RNA genome copied end to end without
loss of sequence
2.Production of (cellular) translationcompetent mRNAs.
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
(-) Strand RNA viruses
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
(-) Strand RNA viruses
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
(+) Strand RNA viruses
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
(+) Strand RNA viruses
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
Ambisense RNA viruses
General strategies for replication
and mRNA synthesis of RNA virus
genomes. (See Fig 6.1)
Double-stranded RNA viruses
RNA-dependent RNA
polymerase (RDRP)
• Unique process, no cellular
parallel
• Hallmark: resistant to
actinomycin D, an inhibitor of
DNA-directed RNA synthesis.
RNA-dependent RNA
polymerase (RDRP)
• Universal rules:
– RNA synthesis initiates and
terminates at specific sites in the
template
– Catalyzed by virus-encoded
polymerases
– Viral and sometimes host accessory
can be required
– (most) can initiate RNA synthesis de
novo (no primer requirement)
• Some do require a free 3’-OH group for
priming
• Primer can be protein linked
– RNA usually synthesized by
template directed, stepwise
incorporation of rNTPs
– Elongation is in 5’  3’ direction
• Examples of non-templated viral RNA
synthesis exist.
• Viral RNA synthesis is highly efficient.
– e.g. Poliovirus RNA copied to 50,000
copies in the course of an 8 hr infection
Three dimensional
structure of RDRPs
• Described as analogous to
a Right Hand with
• Thumb, Palm & Fingers.
• Active site located in the
Palm subdomain
(Fig. 6.3)
Secondary RNA
structures
• First order information content
is contained in the sequence of an
RNA
• Second order information
content is contained the
structure
• Ability to form G-U base pairs, as
well as more exotic non-WatsonCrick base pairs gives RNA the
ability to produce a wide variety
of structures.
• The wide variety of structural
possibilities provides for
specificity of interaction with
other biomolecules, e.g. viral or
host proteins.
Secondary RNA
structures
A wide variety of RNA
structures.
• Stem regions
• Pseuodknots
Each of these can contain
un-paired sections
called loops
• Hairpin loops
• Bulge loops
• Interior loops
• Multibranched loops
Roles of viral accessory
proteins
• Used to direct RDRP to the
correct intracellular site.
– Nucleus – e.g. Influenza
– Membranes – e.g. polio
• Can target RDRP to correct
initiation site on RNA
template
• Helicases unwind RNA
secondary structures
– Processive: unwind along an
mRNA
– Distributive: unwind at one
particular spot
See Figure 6.8 in text
Cellular proteins in viral RNA
synthesis
In the context of viral genome
condensation, viruses have hijacked host
proteins to their service.
• Qb: RDRP requires ribosomal protein S1, EF-Tu
and EF-Ts for their RNA binding properties.
• Poliovirus:
– host-encoded poly(rC)-binding protein 2 helps
target viral proteins to an RNA secondary
structure that is the site of initiation for
genome replication (see. Fig. 6.8)
– Poly-A binding protein 1 (PABP-1) used in
both initiation of replication and translation.
• Cytoskeletal proteins: used in replication
of many RNA viruses. Specific targeting
thought to ensure high local
concentrations of replication components.
– Tubulin: stimulates replication of measles and
Sendai viruses.
– Actin: Human parainfluenza virus type 3,
Respiratory syncytial virus
Initiation Mechanisms
Most initiation occurs de-novo.
Exceptions:
• Protein Priming: Poliovirus VPg
covalently linked to 5’ end of
genome. VPg becomes
polyuridylated (polyU). Base pairs
with polyA 3’ end of genome.
Interaction with RDRP serves to
target replicase to primed 3’ end of
genome. See Fig. 6.8B
• Priming by capped RNA fragments:
Influenza steals 7Methyl-Gppp caps
(cap snatching) from cellular mRNAs
by cleaving cellular mRNAs.
Cleavage products used to prime
viral mRNA synthesis. See fig. 6.9.
The Ribosome/RDRP
clash problem
• In (+) RNA
viruses, RNA is
both template
for translation
and replication.
• Translation
moves in the
5’ 3’ direction
along the (+)
strand
• Replication
moves in the 3’
5’ direction
along the (+)
strand
• Problem: at
some point in
the middle they
will collide.
These viruses
must evolve
around this.
Discrimination between
viral and cellular mRNAs
Q: How do virus RDRPs
discriminate between self
and non-self mRNAs?
A: Through secondary RNA
structures. Called cis-acting
RNA elements.
• Often serve as the switches
between translation and
replication.
Synthesis of polyA
tracts
• 3’ polyA tails are required for
translation of (most) mRNAs
• polyA is attached to the 3’ ends
of cellular mRNAs in the nucleus.
• RNA viruses replicate in
cytoplasm
•  Many RNA Viruses have
evolved mechanisms to acquire
polyA tracts: e.g.
– Encode a 3’ polyA sequence on the
(+) strand and/or 5’ polyU on the (-)
strand
– Reiterative copying (“stuttering”) on
short 3’ U-sequences on the (-)
strand.
Switching from mRNA
production to genome
RNA synthesis
• No switch required when mRNA
and gRNA are identical.
• However, mRNAs of RNA viruses
are not complete copies of the
viral RNA. A switching
mechanism is required.
• Different polymerases for
different functions
– e.g. alphaviruses sequentially
produce 3 RDRPs, each with
template specificity. The last one is
specific for replication of full length
genomic RNA
– e.g. Influenza and VSV viruses
produce two RNA polymerases, only
one of which can produce genomic
RNA
Switching from mRNA
production to genome
RNA synthesis
Different
templates used
for RNA
synthesis and
genome
replication
– e.g. in dsRNA
viruses
replication of
gRNA occurs only
after packaging
RNAs inside of
capsids.
– All unpackaged
viral RNAs are
mRNA by default.
•(fig. 6.17)