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) translation-competent mRNAs.
General strategies for replication and mRNA synthesis of RNA virus genomes.
See Figure 6.1
RNA-dependent RNA polymerase (RDRP)
 Unique process, no cellular parallel
 Hallmark: resistant to actinomycin D, a drug that inhibits DNA-directed RNA
synthesis.
 Universal rules:
o RNA synthesis initiates and terminates at specific sites in the template
o Catalyzed by virus-encoded polymerases
o Viral and sometimes host accessory can be required
o (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
o RNA usually synthesized by template directed, stepwise incorporation of
rNTPs
o Elongation is in 5’  3’ direction
 Examples of non-templated viral RNA synthesis exist.
 Viral RNA synthesis is highly efficient.
o e.g. Poliovirus RNA copied to 50,000 copies in the course of an 8 hr
infection
Three dimensional structure of RDRPs (Fig. 6.3)
 Described as analogous to a Right Hand with
 Thumb, Palm & Fingers.
 Active site located in the Palm subdomain
Secondary RNA structures (see fig 6.7)
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-Watson-Crick base pairs gives
RNA the ability to produce a wide variety of structures. These include:
 Stem regions
 Pseuodknots
Each of these can contain un-paired sections called loops
 Hairpin loops
 Bulge loops
 Interior loops
 Multibranched loops
The wide variety of structural possibilities provides for specificity of interaction with
other biomolecules, e.g. viral or host proteins.
Roles of viral accessory proteins (See fig 6.8)
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
Cellular proteins in viral RNA synthesis
In the context of viral genome condensation, viruses have hijacked host proteins to their
service. Examples:
 Q: RDRP requires ribosomal protein S1, EF-Tu and EF-Ts for their RNA
binding properties.
 Poliovirus:
o host-encoded poly(rC)-binding protein 2 to help target viral proteins to an
RNA secondary structure that is the site of initiation for genome
replication (see. Fig. 6.8)
o 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.
o Tubulin: stimulates replication of measles and Sendai viruses.
o Actin: Human parainfluenza virus type 3, Respiratory syncytial virus
Initiation Mechanisms
Most initiation is 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 (Fig 6.12, and figure from Barry and Miller)
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In (+) RNA viruses, the RNA is both the template for translation and for
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.
Example: the switch between translation and replication modes of Barley
Yellows Dwarf Virus. (See Barry,J.K. and Miller,W.A. (2002). A -1 ribosomal
frameshift element that requires base pairing across four kilebases suggests a
novel mechanism of regulating ribosome and replicase traffic on a viral RNA.
Proc. Natl. Acad. Sci. USA.)
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.
These also 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.
o Encode a 3’ polyA sequence on the (+) strand and/or 5’ polyU on the (-)
strand
o 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
Different templates used for mRNA synthesis and genome replication (fig. 6.17)
 e.g. in dsRNA viruses replication of gRNA occurs only after packaging
RNAs inside of capsids. All unpackaged viral RNAs are mRNA by default.