General Principles De novo initiation RNA polymerase RNA

General Principles • De novo initiation: – RNA polymerase – RNA template – The initiating NTP and a second NTP • Primer Dependant initiation – Protein primer – 5’ Cap – All synthesis begins with the formation of a complex of polymerase, template-primer, and initiating NTPs

The monocistronic m. RNA problem m. RNA AAAA RIBOSOMES PROTEIN AAAA • • Make one monocistronic m. RNA per protein Make a primary transcript and use alternative splicing Make a large protein and then cut it into smaller proteins Include special features in the m. RNA which enable ribosomes to bind internally 3

General Principles • Template specificity – Appears to be very strongly related to : a) b) c) d) 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.

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 m. RNA synthesis and genome replication • Switching from m. RNA to genome replication

6

Positive Strand RNA Viruses

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

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 m. RNA is now available for translation.

Synthesis of viral proteins • Poliovirus virion RNA functions as an m. RNA but does not have the methylated cap structure typical of eucaryotic m. RNAs • 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.

Synthesis of Viral Proteins • • • The m. RNA 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

RNA replication • • • We now have newly made viral proteins to support replication. Viral RNA polymerase copies plus-sense genomic RNA into complementary minussense 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 strands ii. It may be packaged into progeny virions iii. It may be translated into polyprotein (In this case VPg is usually removed prior to translation)

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 [VP 0, 1 and 3]. • VP 0 is then cleaved to VP 2 and VP 4 as the virions mature and these mature virions are infectious. • Virions are released following cell lysis. • Excess capsids are formed and inclusion bodies may be seen in the cytoplasm.

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

Examples of viruses with + sense RNA genomes

Polio Virus

The poliovirus genome

Secondary structure important in viral RNA

Complex Formation on CRE for uridylylation of VPg

• First step is binding of viral RNA genome to membraneous vesicles • Recruited by hydrophobic viral protein that embeds in membrane and anchors viral RNA via an interaction with a host cell protein PCbp. • PCbp binds to RNA cloverleaf structure. • Further interaction of another host protein PAbp allows 3’ end to bind to the coverleaf structure at the 5’ end to form a circle. • The uridylylated VPg is transferred to the 3’ end and serves as a protein primer for RNA synthesis

Alphavirus

The genome

Differences Between Polio and Alphavirus • 5’VPg versus 5’ cap RNA • Translation and production of subgenomic m. RNA in alphavirus.