Regulated translation of bacteriophage mRNA

There is a major difference in the way protein synthesis occurs on bacterial ribosomes as compared to eukaryotic ribosomes, and this leads to a significant difference in the way expression of viral-encoded protein is controlled. As discussed in Chapter 13, bacterial ribosomes can initiate translation at start sites in the interior of bacterial mRNA. This means that a bacterial mRNA molecule with several ORFs can be translated independently into one or all of the proteins. In an RNA bacteriophage infection, protein synthesis programmed by the incoming genome is characterized by synthesis of viral RNA replicase only. Later in infection, after genome replication begins, transition to synthesis of capsid and other proteins begins.

This temporal regulation is governed by the secondary structure of the genome, and initiation of protein synthesis encoded by interior ORFs by ribosomal mechanisms. This can be seen in the phage QP, which is shown as a diagram in Fig. 14.11. This virus encodes three distinct

  1. 14.11 The approximately 25-nm-diameter icosahedral capsid of positive-sense RNA bacteriophage Q^. The positive-sense RNA genome contains three separate open reading frames (ORFs). These ORFs can be independently translated from the full-length virion RNA because unlike the situation in eukaryotic viruses, bacterial ribosomes can initiate translation at interior start signals provided that the ribosome can interact with them. With this bacteriophage, ribosome attachment and translation require active transcription to allow the nascent positive-sense RNA to be unfolded so that the translation start is accessible.
  2. 14.11 The approximately 25-nm-diameter icosahedral capsid of positive-sense RNA bacteriophage Q^. The positive-sense RNA genome contains three separate open reading frames (ORFs). These ORFs can be independently translated from the full-length virion RNA because unlike the situation in eukaryotic viruses, bacterial ribosomes can initiate translation at interior start signals provided that the ribosome can interact with them. With this bacteriophage, ribosome attachment and translation require active transcription to allow the nascent positive-sense RNA to be unfolded so that the translation start is accessible.
Bacteriophage Qp

A protein

Coat

Suppressible stop codon ^ Replicase translational reading frames encoding genes for the A (maturational) protein, the coat protein, and replicase. The coat protein translational reading frame has a translation terminator that is misread (suppressed) as a tryptophan residue about 1% of the time, and when this happens, a larger capsid protein with additional amino acids is generated. Suppression of the termination is absolutely required for phage replication.

A portion of the replication cycle of QP is shown in Fig. 14.12. Ribosomes can associate with the genomic RNA, but this positive-sense genome is folded in such a way that the only start codon available for interaction with a ribosome is the one that begins translation of phage RNA replicase. All other start codons are involved in base-pairing interactions as a part of the

Positive strand RNA

ยก CAC

CAC ,

ZI

t V-X \

Negative strand RNA

Negative strand RNA

Replicase enzyme 3'

'GUG blocked

Fig. 14.12 Coupled transcription-translation of bacteriophage Q^ RNA results in opening the blocked translational start site for the A (maturational) and coat proteins. As the replicase enzyme passes the region containing the translation start site on the negative-sense template (which is a GUG for the A protein), the nascent positive-sense mRNA can interact with a ribosome before it has a chance to fold into a structure in which this initiator codon is sterically blocked. Multiple ribosome entry results in translation of a large number of copies of the maturational and coat proteins being synthesized. High levels of coat protein specifically inhibit translation of replicase from full-length genomic RNA so that replicase is only synthesized at early times in the replication cycle. For this reason, it is often termed an "early" protein or gene product.

secondary structure. For this reason, replicase is the only phage protein expressed at the start of infection.

Synthesis of new positive-sense genomes takes place through formation of RI-1 and RI-2. As new positive-sense genomic RNA disassociates from the negative-sense template near the replicase, secondary structure has not yet formed. This results in the start codon for the A and coat proteins being available to begin translation. The A protein uses a GUG instead of an AUG initiation codon. Similarly, newly replicated positive-sense strands immediately interact with ribo-somes to yield the capsid proteins necessary for the formation of new virus particles.

This simple mechanism ensures that the earliest protein expressed will be replicase. Further, since a relatively large amount of RI-2 will need to be present, synthesis of A and capsid proteins will only occur when there are a large number of genomes waiting to be encapsidated. Multiple entry of ribosomes onto the nascent viral mRNA ensures that a large amount of structural protein will be available when necessary.

Finally, the phage controls the amount of replicase synthesized in infection so that progeny positive-sense strand does not end up recycling too long. Such control is accomplished by the capsid protein actually inhibiting synthesis of replicase from mature positive-sense RNA. Therefore, after about 20 minutes, increasing levels of capsid proteins shut off replicase synthesis.

Case study: enteroviruses

Clinical presentation/case history: Patient is an 18-year-old female who presented with a 2- to 3-week history of upper respiratory symptoms and myalgias (sore or aching muscles) that spontaneously resolved, followed by the development of severe headache, nausea, and vomiting 2 days prior to admission. She presented to the ER where blood tests revealed a high white blood cell count of 38,000/|ll (normal is 4000-12,000/|l). Further case history further revealed that her entire family had also experienced similar symptoms. Her father had also developed a severe headache accompanied by delirium, which had spontaneously resolved. Her twin brothers had also developed similar symptoms accompanied by a rash.

Diagnosis: The patient was admitted for presumptive diagnosis of meningitis, for supportive care, and IV antibiotics. An MRI of the brain showed diffuse, symmetric parenchymal edema of the cortical gray matter and brain stem consistent with meningoencephalitis. In order to differentiate between bacterial meningitis and viral meningitis a spinal tap was performed. Analysis of the CSF revealed no evidence of bacterial antigens, normal levels of glucose, and the presence of neurotrophils. This was consistent with a viral meningoencephalitis. Viral analysis (PCR/RT-PCR) of CSF detected coxsackievirus.

Treatment: There is no treatment for coxsackievirus infections, and only supportive care can be offered. Proper disinfection and handwashing practices are important to prevent transmission of enteroviruses to susceptible individuals.

Disease notes: Enteroviruses are transmitted by the oral-fecal route and are highly infectious. They often cause subclinical or clinically benign cold-like or mild gastrointestinal symptoms. However a number of members of the coxsackievirus genus are associated with a variety of more severe symptoms including infections of the brain (meningitis, encephalitis), infections of the heart (myocarditis and pericarditis), muscle pains that can resemble a heart attack, and hand-foot-and-mouth disease which is a vesicular rash associated with a fever that is common among young children, particularly in daycare settings.

QUESTIONS FOR CHAPTER 14

1 What are the steps in the attachment and entry of poliovirus in a susceptible host cell?

2 The Picornaviridae (e.g., poliovirus) have, as their genome, one molecule of single-stranded RNA. This genomic RNA functions in the cell as a monocistronic mRNA. However, picornavirus-infected cells contain 10 or more viral proteins.

(a) What mechanism have these viruses evolved such that this monocistronic mRNA produces this large number of translation products?

4 The poliovirus genome is a single-stranded RNA of about 7500 nucleotides, with a covalently linked terminal protein, VPg, at the 5' end and a polyA sequence at the 3' end. The polyA tail is not added after replication but is derived from the template during replication. VPg is important for replication of this viral RNA, along with poliovirus polymerase and certain host enzymes.

There are two models for the action of VPg:

Model 1 - VPg may act as a primer for RNA synthesis, being used as VPg-pUOH. Model 2 - VPg may act as an endonuclease, attaching itself to the 5' end of a new RNA chain. In this model, RNA synthesis is primed after addition of U residues to the 3' A at the end of the genome by a host enzyme, followed by a loop-back and self-priming mechanism.

Given these two models, imagine that you have an in vitro system to test the properties of poliovirus genome replication. Your system contains viral genomic RNA as a template and all of the necessary proteins, except as indicated below.

(b) The poliovirus mRNA does not have a 5' methylated cap that is present on host cell mRNA. How do host cell ribosomes begin translation of this message?

3 Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae. Based on your knowledge of the properties of members of this family, complete the following table with respect to FMDV and each of the characteristics listed. State whether the characteristic is present or absent.

  • a) Assume that model 1 is true. What would you expect to see as the product of the reaction if VPg was left out of the mixture?
  • b) Assume that model 2 is true. What would you expect to see as a product of the reaction if endonucleolytic activity of VPg was inhibited?

5 Draw the structure of the poliovirus RI-1 and RI-2. What are the similarities and differences for these two structures?

6 Which of the following statements is (are) true in regards to the poliovirus genome?

  • a) It lacks posttranscriptional addition of repeating adenines.
  • b) It is approximately 1400 bases long.
  • c) It contains a VPg protein that is cleaved prior to packaging.
  • d) It has a single precursor protein that is cleaved by cellular cytoplasmic nucleases.

7 How are the structural proteins of Sindbis virus generated during the infectious cycle?

Characteristic

Present or absent for FMDV

5' methylated cap

Subgenomic RNAs

3' polyadenylation

Single-stranded, positive-sense genome

Expression of genome as a polyprotein

Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Gene Expression

  • REPLICATION OF NEGATIVE-SENSE RNA VIRUSES WITH A MONOPARTITE GENOME
  • Replication of vesicular stomatitis virus - a model for Mononegavirales

Vesicular stomatitis virus virion and genome Generation, capping, and polyadenylation of mRNA Generation of new negative-sense virion RNA Mechanism of host shutoff by vesicular stomatitis virus Cytopathology and diseases caused by rhabdoviruses

  • Paramyxoviruses Pathogenesis of paramyxoviruses
  • Filoviruses and their pathogenesis
  • Bornaviruses
  • INFLUENZA VIRUSES - NEGATIVE-SENSE RNA VIRUSES WITH A MULTIPARTITE GENOME
  • Involvement of the nucleus in flu virus replication
  • Generation of new flu nucleocapsids and maturation of the virus
  • Influenza A epidemics
  • OTHER NEGATIVE-SENSE RNA VIRUSES WITH MULTIPARTITE GENOMES
  • Bunyaviruses

Virus structure and replication Pathogenesis

# Arenaviruses

Virus gene expression Pathogenesis

CHAPTER

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