REGULATION OF TRANSLATION DURING VIRAL INFECTION Interferons are produced in response to viral infection as part of the rapid innate immune response Interferons diffuse to neighboring cells and bind to cell surface receptors to activate transcription of antiviral genes Two interferon induced genes are: RNase L - degrades RNA protein kinase RNA-activated (Pkr)- phosphorylates eIF2 , inhibiting translation initiation

Pkr is a serine threonine kinase composed of an N-terminal regulatory domain and a C- terminal catalytic domain Pkr is activated by the binding of dsRNA to two dsRNA binding motifs at the N-terminus of the protein. Activation leads to autophosphorylation of Pkr dsRNA activated protein kinase (Pkr)

Phosphorylation of eIF2  inhibits recycling of eIF2

Model of activation of Pkr

Viral regulation of Pkr Viruses use at least five different mechanisms to block Pkr activation or to stop activated Pkr from inhibiting translation inhibition of dsRNA binding- adenovirus VA RNA binds Pkr blocks its activation by dsRNA binding and sequestering dsRNA- vaccinia virus E3L protein inhibition of Pkr dimerization- hepatitis C virus NS5A inhibitors of kinase function- vaccinia virus K3L protein has homology to N-terminus of eIF2- 

Regulation of eIF4F activity by different viruses 1) Phosphorylation of eIF4E 2) Cleavage of eIF4G Rapamycin

Regulation of poly A binding protein activity Poly A binding protein interaction with eIF4G, bring together the ends of the mRNA Rotaviruses inhibit host translation by blocking the function of Pab1p Viral protein nsP3 occupies the binding site of Pabp1p on eIF4G It prevents binding of Pab1p to eIF4G preventing formation of the circular complex, allowing translation of viral RNAs

What a Typical Virus Must Do to Survive  Find the right cell and enter the cell.  Uncoat to activate the viral genome.  Translate the viral genes.  Replicate the viral genome.  Assemble new virus particles.  Exit from the cell.  Find a new host to infect. Every virus must be able to exploit specific processes of its host for gene expression and replication, and must be able to overcome host defenses.

Genomes of RNA viruses:  unimolecular  segmented  single stranded of (+) polarity  single stranded of (-) polarity  double stranded  circular Common requirement: They must be copied within the infected cell to provide new genomes and mRNAs

Genomes of RNA viruses The genomes of all RNA viruses except retroviruses encode an RNA-dependent RNA polymerase to Catalyze the synthesis of new genomes and mRNAs RNA viruses with (-) strand and double stranded RNA genomes must contain the RNA polymerase RNA viruses with (+) strand RNA genome lack a virion polymerase Questions: Are RNAs (-) strand viruses infectious? Are RNAs of (+) strand viruses infectious?

 Plus-strand RNA viruses do not have a polymerase in their virions. RNAs of these viruses are infectious, but they must first be translated in cells to produce a viral RNA-Dependent-RNA- Polymerase (RDRP) is synthesized for genome replication.  After the genomic RNA is copied into a negative strand, it serves as a template for replication of progeny plus-strand RNAs. There is no DNA phase to replication of these viruses.  Picorna-like viruses, such as Poliovirus and some plant viruses, that express their genomes by proteolytic processing translate their genomes as large polyproteins from their genomic RNAs that is processed into individual viral proteins.  Alpha-like viruses, such as sindbis virus and tobacco mosaic virus synthesize subgenomic mRNAs from the negative strand, which contains promoters that are recognized by the replicase. Replication and Expression of RNA Virus Genomes

Replication of Plus-Sense Viral RNA (-) (+) (-) (+) * * * * (-) * * * * (+) Genomic RNA (+ssRNA) or Synthesis of Minus-Strand By RDRP Synthesis of Progeny Plus- Strand RNA By RDRP Replicative intermediate Replicative Form I Replicative Form II Synthesis of Minus-Strand Intermediates ….. Intermediates are Double-stranded RNA; Not DNA Intermediates. No Proof Reading as in DNA replication so replication is error prone.

Plus Strand RNA Viruses Replicate in the Cytoplasm  After Plus Strand RNA viruses enter cells, the next in step replication is release of the genomic RNA.  The viral RNA must then be translated for expression of the RNA-Dependent-RNA Polymerase (RDRP).  The RDRP functions in transcription of mRNAs and replication of genomic RNAs.  Viral capsid proteins are translated and assemble in the cytoplasm with the viral RNA.  Unenveloped viruses are released by lysis, enveloped viruses bud from the plasma membrane and are released from the cell. Several Variations of this theme Occur

Nonsegmented Negative Strand RNA Viruses  Minus sense RNA genomes are complementary to their mRNAs.  Viral genomic RNA not infectious.  All negative strand viruses are enveloped.  Virus particles have nucleocapsid cores consisting of the minus- sense genomic RNA encapsidated by core-associated proteins. mRNAs Genomic RNA Antigenomic RNA Many Progeny Genomic RNAs Negative strand RNA Replication  Nucleocapsids have RNA-dependent- RNA polymerase activity.  Polymerase transcribes the Viral genomic RNA into mRNA and plus-sense antigenomic RNA.  Antigenomic RNAs are copied into progeny minus-sense genomic RNAs.  Progeny genomic RNAs synthesizemore mRNAs and function to form virion formation.

Generalized Replication Strategy of Negative Strand Viruses 1) Virus enters cell by endocytosis. 2) The viral nucleocapsid core is released. 3) Viral mRNAs are transcribed in the core from the negative strand genome. 4) Viral proteins are translated & accumulate in cells. 5) As proteins increase in amount they associate with the newly synthesized plus sense RNAs to form antigenomic nucleocapsids. 6) The antigenomic RNAs replicate to form progeny genomic (minus sense) nucleocapsids. 7) New rounds of mRNA synthesis occur and replication cycles are repeated. 8) The minus sense nucleocapsids undergo morphogenesis and virus is released from cells.

Life Cycle of Double Stranded RNA Viruses 1) Viruses with dsRNA genomes are complex, contain several segments, and an RNA-dependent- RNA-Polymerase. 2) Particles enter cells via endocytosis. 3) Proteolytic digestion results in subviral particles. 4) Core particle moves into the cytoplasm & begin synthesis of early viral mRNAs from the dsRNA genomic RNAs. 5) Late or secondary mRNA transcripts begin to appear at six to eight hours post infection, suggesting that an early protein is required for secondary mRNA synthesis. 6) The mRNAs appear to be translated & assemble with viral proteins to form the early nascent cores. 7) Double stranded RNAs are synthesized in the cores, which undergo a series of steps to form mature virus particles that are released from the cell. A key point is that all mRNA transcription and genomic RNA replication occurs in the cytoplasm in viral cores.

Reverse transcription is the hallmark of the retrovirus replication cycle. Viral Mediated Events: Virus enters cells by direct fusion or endocytosis. Icosahedral viral particle is released into the cytoplasm and begins to transcribe double stranded DNA from the diploid RNA genome. An integration complex is transported to the nucleus and functions to integrate the viral DNA into the host genome. Host mediated Events: The integrated viral DNA is transcribed by host RNA polymerase II to produce full length viral RNAs. These RNAs are differentially spliced to produce viral genomic and mRNAs and are exported to the cytoplasm. Viral proteins are translated and assemble to form virions. Retrovirus Replication Cycle

Generalized Replication Strategy of DNA Viruses 1) Virus enters cell and DNA is released. 2) DNA moves to the nucleus and early mRNAs are transcribed using host DNA dependent RNA polymerase. 3) Early mRNAs are translated and proteins elicit progeny DNA replication. 4) Late mRNAs are synthesized to produce capsids for assembly of progeny DNA. 5) Encapsidated DNA exits the nucleus and is released by lysis of host cells. Note: Many different strategies of DNA replication have been identified and several different DNA virus families exist. Viral DNAs do not integrate into the host genome during lytic replication, but may during abortive infections.

Strategies for replication of RNA viruses:

RNA Dependent RNA Polymerase (RDRP):  first evidence in 1960s with mengovirus and poliovirus  can synthesize viral RNA in the presence of actinomycin D  poliovirus 3Dpol can copy polyadenylated genomic RNA in the presence of an oligo (U) primer and ribonucleoside triphosphates (ATP, UTP, CTP and GTP)  many RdRps are associated with membranes or nucleocapsids in infected cells  some require a primer, others can initiate RNA synthesis without a primer

RNA-directed RNA synthesis follows universal rules:  RNA synthesis initiates and terminates at specific sites on the viral RNA template  Catalyzed by virus encoded polymerase, but viral accessory proteins and host proteins may be required  Some require a primer with a free 3’ –OH end to which nucleotides complementary to the template strand are added  RNA primers may be protein linked or may contain a 5’ cap  RNA is synthesized by template directed incorporation of NMPs into 3’ end

Common motifs have been identified in the Sequences of all RNA polymerases Motif C includes the Gly-Asp-Asp sequence (GDD) conserved in RNA polymerases of Most (+) strand RNA viruses part of the active site of the enzyme

Poliovirus genome organization Poliovirus replication occurs on membranes. 2C and 3AB bring the RNA polymerase to membranes. 2C anchors viral RNA to membranes during replication. 3AB anchors the viral primer, VPg to membranes. 3Dpol is brought to the replication complex by binding to 3AB

Poliovirus replication: Polioviral RNA is linked to VPg via a tyrosine. This bond is cleaved by a cellular enzyme to produce viral mRNA containing a 5’ terminal Up.

Minus strand synthesis: 5’ end of polioviral RNA contains a cloverleaf structure and the 3’ end contains a pseudoknot Precursor of VPg, 3AB, acts as a VPg donor A ribonucleoprotein complex is formed when PCbp and 3CDpro bind to the cloverleaf structure This complex interacts with PAbp1, producing circular genome

Protease 3CDpro cleaves membrane bound 3AB to produce VPg VPg is uridylated by 3Dpol and transferred to the 3’ end of the genome 3D pol uses uridylated VPg as a primer for (–) strand RNA synthesis

Plus strand synthesis: The strands of the RF are separated by 2C, which binds to cloverleaf in the (-) strand Membrane bound 3AB is cleaved to produce VPg Uridylated VPg is synthesized by 3D pol, using (–) strand RNA as a template Uridylated VPg is then elongated by 3D pol to Synthesize (+) strand RNA

Host factors required for poliovirus replication  Poly(rC) binding protein 2- Binds to cloverleaf structure at the 5’ end. Formation of the 5’ cloverleaf, 3CD pro and poly(rC) binding protein is necessary for initiation of viral RNA synthesis.  Poly(A) binding protein 1-interacts with poly(rC) binding protein 3CDpro and 3’ poly(A) tail, circularizing the genome.

Imbalance of (-) and (+) strand synthesis:  In poliovirus infected cells, genomic RNA is produced at 100-fold higher concentration than the (-) strand RNA  Ribosomes must be cleared from RNA before it can serve as a template for (-) strand synthesis