Question

Question: How does poliovirus replicate in the epithelial cell of intestine? Describe in detail, not only main idea.

Answer 1

Poliovirus is a small, nonenveloped icosahedral virus of about 30 nm in diameter. It possesses a 7.5 kb +ssRNA genome that is infectious, translated immediately upon entry into the cell. The genome contains a 5′-nontranslated region (NTR) that contains an internal ribosome entry site (IRES), followed by a single open reading frame encoding a polyprotein that is cleaved sequentially into intermediate precursors and 11 mature poliovirus proteins. The genome is also polyadenylated at the 3′-NTR. A viral protein known as VPg (virion protein, genome-linked)associates with the 5′-end of the genome and is thought to act as a protein primer for replication of the genome.

1 Attachment, Penetration, and Uncoating:

The poliovirus capsid is composed of 60 copies each of 4 repeating proteins—VP1, VP2, VP3, and VP4. VP2 and VP4 are derived from autocatalysis of VP0 during maturation of the virion. VP1, VP2, and VP3 form the surface of the capsid, with VP4 associating on the inner capsid wall. VP1 forms a star-shaped plateau or “mesa” on the fivefold axis, which is surrounded by a deep canyon into which the cell surface receptor binds.

The poliovirus virion is acid resistant, which allows it to survive the low pH of the stomach to initiate infection within the small intestine. The cell surface receptor for all three poliovirusserotypes is CD155, a glycoprotein that functions as an adhesion molecule in adherens junctions. In addition, CD155 is also recognized by NK cells to induce their cytotoxicity. CD155 is also commonly referred to as the “poliovirusreceptor,” or PVR. It is expressed on the surface of intestinal epithelial cells and on M cells of Peyer’s patches, which may facilitate their entry into the Peyer’s patches following infection of the intestinal epithelium.

CD155 is not expressed in rodents and small mammals, which explains why experiments that attempted to infect these animals have historically been unsuccessful. In 1990, a transgenic mouse strain was engineered to express the human CD155 molecule. These mice were susceptible to infection, whereas the normal nontransgenic mice were not.

Upon interaction with CD155, the poliovirus capsid undergoes a conformation change whereby VP1 inserts into the cell membrane, forming a pore through which the viral genome is released. This was initially thought to occur at the plasma membrane, although now evidence suggests that this occurs after internalization of the virion by clathrin- and caveolin-independent endocytosis.

2 Translation and Replication:

Within the cytoplasm, VPg is removed by a host DNA repair enzyme, TDP2, often referred to as “VPg unlinkase.” The IRES found at the 5′-end of the +ssRNA genome recruits cellular proteins that direct the assembly of translation initiation complexes, and ribosomes translate the single polyprotein of ∼3000 amino acids in length (∼250 kD). The polyprotein undergoes several cleavages to produce hybrid and individual proteins of various functions. It is first divided into three precursors: P1, P2, and P3. P1 comprises the capsid proteins, and P2 and P3 contain the nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp) 3Dpol, viral proteinases 2Apro, 3CDpro, and 3Cpro, VPg (3B), and other proteins necessary for replication. Interestingly, several of the intermediate polypeptides have very important functions. Notably, 3CDpro is one of the major viral proteinases.

The importance of the IRES in poliovirus virulence is apparent when examining the differences between wild poliovirus and the attenuated OPV strains. Sequencing of these genomes has shown that point mutations in the IRES of each attenuated strain cause defects during translation within neuronal cells, possibly resulting in a strain with reduced neurovirulence.

As a +ssRNA virus, translation of viral proteins precedes replication of the genome in order to produce the RdRp, called 3Dpol. Like all picornaviruses, poliovirus induces the formation of replication complexes (RCs), membrane vesicles derived from the endoplasmic reticulum on which arrays of RdRp form for genome replication. The poliovirus VPg functions as a primer for synthesis of both negative- and positive-strand RNAs. For synthesis of the negative-sense antigenomic RNA, the RdRp adds two uracil-containing nucleotides to VPg, using the poly(A) tail at the 3′-end of the +ssRNA genome as a template. It uses VPg-pU-pU as a primer to copy the genomic RNA into −ssRNA, which functions as a template for genome replication and transcription of additional mRNAs.

For synthesis of the +ssRNA genome, VPg is again used as a primer, except that it adds the two uridine triphosphates using a cis-acting replication element (CRE) as a template. CREs are sequences that are found within the coding regions of the poliovirus genome. They form secondary stem-loop structures that bind viral proteins that assist the RdRp in identifying its viral RNAs from among the polyadenylated cellular mRNAs.

3 Assembly, Maturation, and Release:

The 2Apro cleaves the single poliovirus polyprotein between P1 and P2 to release the P1 precursor, which contains all components of the capsid. The P1 precursor is cleaved by 3CDpro to release VP1 and VP3 proteins, along with VP0, an immature protein that will later be cleaved into VP2 and VP4 as part of the maturation process. VP1, VP3, and VP0 associate with each other to form the structural units, known as protomers. Five protomers spontaneously aggregate to form a pentamer, and twelve pentamers (for a total of 60 structural units) form the procapsid.

The +ssRNA genome and covalently associated VPg are packaged into the procapsid, or alternatively, the procapsid assembles around the genome. Virions undergo maturation into an infectious virion upon the cleavage of VP0 into VP2 and VP4 by what is thought to be a host protease. Poliovirus has been considered a lytic virus, although evidence has emerged recently that in certain conditions release can also occur in a nonlytic fashion through the release of virus within vesicles that have been hijacked from those involved in autophagy, a process that degrades and recycles damaged intracellular components, including organelles.