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Objectives

Objectives. To understand the general principles involved in RNA replication discussed in Chapter 6 pages 175-192 To use the following + stranded RNA viruses as examples and to understand the different processes that during replication by RNA viruses:- Picornavirus Togaviruses Flaviviruses.

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Objectives

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  1. Objectives • To understand the general principles involved in RNA replication discussed in Chapter 6 pages 175-192 • To use the following + stranded RNA viruses as examples and to understand the different processes that during replication by RNA viruses:- • Picornavirus • Togaviruses • Flaviviruses

  2. General Principles • De novo initiation: • RNA polymerase • RNA template • The initiating NTP and a second NTP

  3. General Principles • Primer Dependent Initiation • Protein priming • Capped RNA fragments • All synthesis begins with the formation of a complex of polymerase, template-primer, and initiating NTPs

  4. General Principles • Template specificity • Appears to be very strongly related to : • Affinity of RNA polymerase for the initiating NTP • The sequence • The structure of the viral RNA molecules • Encoding proteins that bind to genomic RNA segments which then activates an endonuclease that cleaves host cell RNAs.

  5. More facts • Unwinding the RNA template • Encoded in genome of ds RNA viruses • Prevent base pairing between template and new strand. • Role of host cell proteins • Different RNA polymerases for mRNA synthesis and genome replication • Switching from mRNA to genome replication

  6. Positive Strand RNA Viruses

  7. PICORNAVIRUSES   (PICORNAVIRIDAE) • Properties • These are small (28nm), • naked • icosahedralviruses • RNA is single-stranded, plus sense, polyadenylated. • functions as mRNA immediately upon infection • E.g poliovirus

  8. Adsorption and penetration • A viral protein recognizes a receptor on the host cell membrane (this is important in the tropism of virus).It seems that binding to the receptor alters capsid structure in some way, a channel forms across the cell membrane and the RNA is released into cytoplasm. The mRNA is now available for translation.

  9. Synthesis of viral proteins • Poliovirus virion RNA functions as an mRNA but does not have the methylated cap structure typical of eucaryotic mRNAs • It has a "ribosome landing pad" (known as the internal ribosome entry site or IRES) which enables ribosomes to bind without having to recognize a 5' methylated cap structure • Most host cell translation is cap-dependent, so this inhibits a lot of host protein synthesis but not viral protein synthesis. 

  10. Synthesis of Viral Proteins • The mRNA is translated into a single polypeptide (polyprotein), which is cleaved. • The cleavages occur before translation is complete ( i.e. on the nascent (=growing) chain) and are carried out by virally coded proteases. • Products of cleavage include: • An RNA polymerase (replicase) • Structural components of the virion • Proteases

  11. RNA replication • We now have newly made viral proteins to support replication. • Viral RNA polymerase copies plus-sense genomic RNA into complementary minus-sense RNA requiring: • VPg (Viral Protein genome-linked) • Viral RNA polymerase (replicase) • Certain Host proteins • VPg may act as a primer for RNA synthesis, this would explain why it is at the 5' end of all newly synthesized RNA molecules • New minus sense strands serve as template for new plus sense strands • Again, poliovirus RNA polymerase and VPg are needed. VPg is linked to the 5' ends of the new plus sense strands (again, it probably functions as a primer). • The new plus strand has three alternative fates:i. It may serve as a template for more minus strandsii. It may be packaged into progeny virionsiii. It may be translated into polyprotein (In this case VPg is usually removed prior to translation)

  12. Assembly • When sufficient plus-sense progeny RNA and virion proteins have accumulated, assembly begins. • Particles assemble with VPg-RNA inside and 3 proteins in the capsid [VP0,1 and 3]. • VP0 is then cleaved to VP2 and VP4 as the virions mature and these mature virions are infectious. • Virionsare released following cell lysis. • Excess capsids are formed and inclusion bodies may be seen in the cytoplasm.

  13. NOTE: THE ENTIRE LIFE CYCLE OCCURS IN THE CYTOPLASM • THERE IS NO DIVISION INTO EARLY AND LATE GENE EXPRESSION

  14. NON-SEGMENTED NEGATIVE STRAND VIRUSES

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