Reading_ViralGeneExp_1 - Regulation of Viral Gene...

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Unformatted text preview: Regulation of Viral Gene Expression & Antiviral Drug Targets Viruses regulate gene expression during lytic phases of growth to achieve the appropriate ratio and timing of gene expression. Structural proteins, such as coat proteins, are needed in higher quantities than enzymatic proteins, such as the replicase protein. Functions involved in lysis have to be expressed late in the lytic cycle to avoid lysis of the cells before new vial particles are assembled. The particular methods used by viruses to ensure proper levels and timing of gene expression vary depending on the nature of their genome, the cell type infected, and the intracellular location of the viral genome in the case of eukaryotic cells. Some of the strategies used by viruses to regulate gene expression are also used by host cells. ssRNA (+) Genomes Differential Translation Efficiency-Prokaryotic Viruses The small MS2 (ssRNA + strand) genome codes for 4 proteins (see figure 1): maturation protein (a protease), coat, lysis protein, and replicase. The MS2 genome is translated on entry into a host cell (Escherichia coli) to generate replicase for synthesis of a negative-strand RNA and more positive-strands from this negative strand template. The MS2 genome folds into a complicated RNA structure. Of the four genes, only the ribosome-binding sites (RBS) and start codons for the coat protein and replicase are easily accessible to the ribosome. Once the coat protein accumulates in the cell, it binds to the RBS region for the replicase and prevents translation of the replicase. The complicated structure of the RBS region of the maturation protein limits translations of this protein. The lysis gene overlaps the 3’ end of the coat protein gene and the 5’ end of the replicase gene. This leads to efficient usage of the viral genome. The lysis RBS is folded into a complicated structure that is relieved by termination of ribosomal translation of the coat protein. How this leads to low translation levels of the lysis protein relative to the coat protein is not fully understood. Figure 1. From: Brock Biology of Microorganisms 10th Edition. The points to be remembered from this text: Know how RNA folding can limit the expression of a gene. Know how higher levels of the coat protein are achieved relative to the maturation protein. Know how higher levels of the coat protein are achieved relative to the replicase. Know how the RNA folding that is occluding access of the ribosome-binding site of the lysis protein is overcome to allow occasional production of the lysis protein. 1 Be familiar with the concept of overlapping genes. Cleavage of a Polyprotein-Eukaryotic Viruses NOTE: Remember that, for translation to happen in eukaryotic cells via the host cell ribosome, RNA must have a 5’-cap and a polyA-tail. The 5’ end of the ssRNA(+) genome of polivirus is covalently liked to a protein and folds into several stem loops. This structure mimics the Capbinding complex and allows the poliovirus genome to binding to ribosomes. Figure 2. Cap-mimic. From: Figure 3. Polyprotein cleavage. From: Textbook Reading Pages 412-413 “Poliovirus” The points to be remembered from this text: Know how multiple proteins can be produced from one larger protein. ssRNA (-) Genomes Segmented Genome and Alternative Splicing The example here is influenza virus. The genome is multiple small ssRNA(-) pieces. Each ssRNA(-) molecules codes for a different protein. The ssRNA(-) must be transcribed into mRNA (Fig. 4). To ensure that mRNA has a 5’ cap, the virus encodes an endonuclease that cleaves near the 5’ end of cellular mRNAs. These 5’ ends of the cellular RNAs serve as primers for synthesis of the viral mRNAs using the viral RNA polymerase. 2 Figure 4. Hijacking cellular 5’-caps. From: Some of the viral mRNAs code for two different proteins. This is accomplished by alternative splicing (Fig. 5). The mRNA has two alternative 5’ splice sites (5’ ss) and one 3’ splice site. At early times in infection, the first 5’ ss is used. At later time points during infection, proteins bind to the mRNA blocking the first 5’ss. The spliceosome then binds to and uses the second 5’ ss. Figure 5. From: Proc Natl Acad Sci U S A. 1995. 92(14):6324-8 The points to know from the influenza virus example: Know that the genome of the influenza virus contains 8 separate pieces of ssRNA(-). This is a segmented genome. Know that a viral endonuclease cleaves cellular mRNAs to release a 5’capped RNA that serves to prime synthesis of mRNA from the ssRNA(-) 3 viral genome (see Figure 4). Know that six of the eight RNA molecules can code for one protein and that two of the RNA molecules code for two proteins via alternative splicing (see Figure 5). Transcription into Monocistronic mRNAs “Following attachment of VSV to the surface of a cell, the genome of VSV is delivered into the cytoplasm. RNA synthesis occurs entirely in the cytoplasm, and the steps that occur are depicted in Fig. 6. Initially, in a process referred to as primary transcription, the RdRP responds to specific signals in the template ssRNA(-) genome to transcribe six discrete RNAs: a 47nucleotide leader RNA (Le+), that is neither capped nor polyadenylated, and 5 mRNAs that are capped and methylated at the 5′ end, and polyadenylated at the 3′ end. Following translation of the mRNAs to yield the viral proteins, genome replication can begin. During replication, the RdRP initiates at the extreme 3′ end of the genome, ignores all the signals for production of discrete monocistronic mRNAs and instead synthesizes a full-length complementary antigenome (i.e. ssRNA(+). In turn, the antigenomic RNA serves as template for synthesis of a 45-nucleotide minus sense leader RNA (Le−), and also for synthesis of full-length progeny genomes (i.e. ssRNA(-). These genomes can then be utilized as templates for secondary transcription, or assembled into infectious particles. What determines whether an RdRP will function as a transcriptase or a replicase is poorly understood, although availability of soluble N protein to encapsidate the nascent genomic and antigenomic RNA is thought to be a key factor.” Figure 6. VSV genome organization and the products of VSV transcription and replication. Shown 3′ to 5′ are the 3′ 50-nt leader region (le), and the genes encoding the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L), respectively, followed by the 5′ trailer region (tr). The products of transcription, the leader (Le+) and the five capped and polyadenylated monocistronic mRNA are indicated diagrammatically below the genome in their relative, respective, transcriptional abundance. The full-length antigenomic positive-strand RNA product of genomic replication is shown above the template. This positive strand antigenome subsequently serves as the template for abundant negative-sense genome synthesis. The ends of the positive strand are labeled, leC=complement of the genomic leader region, trC=complement of the genomic trailer region. From: Biochimica et Biophysica Acta 2002, Pages 337-353. 4 ...
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