How Does Poliovirus Replicate?

Below is my overview of the replication cycle of poliovirus. I've included references in case you want further information. Poliovirus is a common virus studied in scientific labs, and its replication process will fascinate you.

The poliovirus positive sense RNA genome is flanked by a pseudoknot and a region of polyadenylation at its 3’ end and by a VPg, a cloverleaf structure, and an internal ribosomal entry site (IRES) and at its 5’ end (Flint et al, 2000). The 5’ cloverleaf and the IRES are both conserved regions of secondary structure. The 5’ cloverleaf binds cellular and viral factors to regulate genome replication and maintain RNA stability (Lyons et al, 2001), whereas the IRES is responsible for associating with factors which recruit the 40S ribosomal subunit during translation initiation. The polioviral genome is organized into three primary regions, denoted P1, P2, and P3. P1 codes for the coat precursor, while P2 and P3 code for nonstructural proteins involved in proteolytic processing and synthesis. A conserved stemloop structure called the cis-regulatory element (CRE) has recently been mapped to the genome’s center and is thought to facilitate communication between the 5’ and 3’ ends (Murray and Barton, 2003). Poliovirus conducts its entire replication cycle within the cytoplasm of a permissive eukaryotic host cell (Fields et al, 1996).
Immediately after entry, the poliovirus genome is translated by host cell factors into a single polyprotein. Translation initiation occurs at the IRES, where viral factors and cellular trans acting factors (ITAFs) associate and recruit ribosomes (Boussidia et al, 2003). The polyprotein is cleaved numerous times to ultimately generate eleven gene products. The first cleavage, which releases P1 from P2 and P3, is autocatalytic and occurs while the nascent protein is being translated (Flint et al, 2000). During later stages of infection, this cleavage is also catalyzed by 3CD, a product of P3, and by 2Apro, a product of P2 (Fields et al, 1996). P1 is sub-cleaved into the capsid proteins VP1, VP3, and the capsid precursor VP0. P2 is self-cleaved to release the protease 2Apro. The P2 region also contains 2BC, a precursor protein which inhibits cellular transport. 2BC gives rise to 2B and 2C. 2B is thought to be a host range determinant and a cellular transport inhibitor, and 2C functions as a 3Dpol transporter and an RNA anchor during negative strand synthesis (Cuconati et al, 1998). The P3 region can either be self-cleaved, or can be processed by its own cleavage product, 3Cpro. As with P1, self-cleavages occur during initial infection stages, when protease concentrations are limiting. P3 products include the precursor proteins 3AB and 3CD. 3AB assists 2C during 3Dpol transport, and gives rise to 3A, which inhibits cellular transport, and 3B, which is also known as VPg (Fields et al, 1996). 3CD mainly functions as a protease, but is also sub-cleaved to generate 3Dpol and 3Cpro. 3Dpol –the viral RNA-dependent-RNA-polymerase (RdRp)—is a primer- and template-dependent replicase which creates an opposite polarity RNA molecule from a positive- or negative-strand viral template. Poliovirus cannot replicate its genome until it translates 3Dpol because the host cell’s machinery is not equipped to replicate RNA (Flint et al, 2000). Poliovirus avoids the problem of translating a polycistronic genome with eukaryotic cellular machinery by synthesizing a single multifunctional polyprotein and generating active gene products by cleavage. Despite this benefit, poliovirus cannot regulate gene expression using this mechanism. Consequently, nonstructural gene products such as proteases and 3Dpol must be generated at higher levels than are necessary in order to maintain adequate levels of capsid proteins (Flint et al, 2000).
Within in thirty minutes of poliovirus infection, 2Apro cleaves the host eukaryotic initiation factor 4G (eIF4G) (Koch and Koch, 1985). eIF4G is a subunit of the cap binding complex which functions in ribosomal recognition of the 5’ 7-methyl guanosine cap and subsequent translation of host cell mRNA. When eIF4G is cleaved, formation of the cap binding complex is inhibited, and translation of host mRNAs is precipitously reduced. This increases the concentration of free ribosomes for viral mRNA translation and ultimately stimulates membrane permeabilization and cell breakdown, allowing for cell lysis and virion exit. Cleavage of eIF4G also releases its C-terminal region which is required for recruitment of ribosomal 40S subunits to the viral IRES. Thus, 2Apro simultaneously inhibits cellular protein synthesis and stimulates viral protein synthesis through a single processing event (Fields et al, 1996).
Within three hours post-entry, poliovirus stimulates vesicle proliferation by inhibiting transport from the endoplasmic reticulum (ER) to the cell surface (Koch and Koch, 1985). Vesicles radiate outward from the nuclear envelope, essentially packing the host cell. Poliovirus requires these vesicular membranes to anchor cellular factors during viral RNA synthesis and packaging. Additionally, tight membrane associations may compartmentalize high local concentrations of necessary molecules and increase reaction rates and efficiencies (Teterina et al, 2001). The resulting cytopathic effect is a significant rearrangement of internal cellular membranes and a disruption of nuclear RNA export. Additionally, the infected cell cannot transport cell surface antigens and is therefore less likely to be detected by the immune system. These events greatly compromise the cell, but do not adversely affect the virus which has no use for glycosylation or transport because it is nonenveloped (Flint et al, 2000).
During early stages of infection, poliovirus seems to reroute cellular transport. The protein Sam68 (Src-association in mitosis, 68kD) migrates from its normal nuclear environment and associates with 3Dpol in the cytoplasm. Similarly, the nucleolar protein, nucleolin, moves to the cytoplasm and interacts with the viral genome to increase efficiency of genome synthesis during early stages of infection (Waggoner and Sarnow, 1998). These nuclear proteins may shuttle between the cytoplasm and nucleus and be trapped in the cytoplasm after viral inhibition of vesicular transport occurs. Alternatively, the proteins may be used as chaperones, recruited by the virus while viral protein concentrations are limiting (Waggoner and Sarnow, 1998).
Once viral protein concentrations are sufficient and vesicle formation has occurred, poliovirus replicates its positive strand RNA genome into its negative strand complement. The proposed mechanism for negative strand synthesis includes the transport of 3Dpol to the vesicular replication site, which is mediated by 2C and 3AB (Flintet al, 2000). 3CD and poly(rC) binding protein (PCBP), a cellular ITAF, associate with the 5’ cloverleaf of the template RNA. This mechanism may commit the template to synthesis, or prevent the formation of a translation initiation complex (Teterina et al, 2001; Lyons et al, 2001). Additionally, crosstalk between the 3’ and 5’ ends of the template is required to initiate replication (Teterina et al, 2001). In this model, the genome circularizes when 3CD proteins at the 3’ and 5’ ends dimerize (Murray and Barton, 2003). Crosstalk may be a mechanism for coordination of negative strand synthesis and translation (Lyons et al, 2001). Following formation of these initiation complexes, 3AB becomes anchored within the host membrane and 3CD mediates its polyuridylation by transferring free uracil monophosphates (UMPs). 3AB is then cleaved by 3CD to release VPgpUpUOH. VPgpUpUOH then binds to the polyadenylated 3’ pseudoknot region of the positive strand genome and serves as a primer for extension by 3Dpol (Arnold and Cameron, 2000). The negative strand is subsequently used as a template to generate numerous positive strands. As many as eight “daughters” can be transcribed simultaneously through this replicative intermediate which can support multiple, continuous cycles of initiation (Arnold and Cameron, 2000).
Recent literature has contradicted this traditional model for negative strand synthesis by reporting that VPg uridylation is only required in positive strand synthesis, and that the tyrosine-OH residue of VPg is sufficient to prime negative strand synthesis (Murray and Barton, 2003). Moreover, in the proposed model of positive strand synthesis, a primer is created by 3Dpol-mediated VPg uridylation, using the CRE as a template (Murray and Barton, 2003). Apart from these key differences, positive strands are thought to be synthesized through the same pathway as negative strands (Fields et al, 1996).
When genome replication is complete, the ratio of positive- to negative- strand RNA is greater than 30:1 (Barton et al, 1995). A proliferation of positive strands is useful because the positive genome has many roles in polioviral replication. It can serve as a template for translation or negative strand synthesis, and it is also encapsidated as the genome. The fate of each positive strand must therefore be regulated to prevent problems such as collisions between ribosomes and 3Dpol, and also to prevent encapsidation of negative strands (Fields et al, 1996). This regulation has been confirmed by mutational analysis, and is thought to be temporal and concentration-dependent. As the concentration of capsid proteins increases, most positive-strand RNAs are encapsidated rather than undergoing translation or negative strand synthesis. Capsid protein concentration is similarly regulated by 3CD concentration because 3CD is capable of cleaving P1, the capsid protein precursor, from the polyprotein (Flint et al, 2000). It has also been proposed that only RNA molecules actively undergoing replication are stably anchored to vesicles, and only newly-replicated positive strand molecules are encapsidated (Teterina et al, 2001; Nugent et al, 1999). Alternatively, RNA molecules destined for translation are released into the cytoplasm. Taken together, these results support a model in which replication and encapsidation are coupled at vesicular membranes and are sequestered from translation initiation (Johansen and Morrow, 2000). Additional levels of positive sense RNA regulation have also been proposed. The 5’ cloverleaf and the IRES have been implicated as a regulatory “switches” between replication/encapsidation and translation. High concentrations of 3CD bind to the cloverleaf and repress translation in favor of encapsidation or replication (Johansen and Morrow, 2000). Alternatively, ITAFs may serve as chaperone proteins, folding the IRES into a secondary structure which promotes translation and signals release of the RNA (Boussidia et al, 2003).
Assembly and encapsidation occur within four to six hours post-entry after virion proteins have reached sufficient concentrations and the viral genome has been replicated (Koch and Koch, 1985). As the concentrations of P1 and therefore of VP1, VP3, and VP0 increase, pentamer assembly becomes favorable. Sets of twelve pentamers associate to form procapsids into which positive-strand VPg-bound RNA is encapsidated to form the provirion (Flint et al, 2000). Each association step in the pathway is stabilized by extensive protein interactions within the developing capsid, and is thought to be mediated by host cell chaperones, such as heat shock proteins. It is unknown whether pentamers condense around genomes or whether genomes are threaded into preexisting procapsids. The final “maturation cleavage” of VP0 to VP2 and VP4 follows encapsidation and creates a state of metastability which is required to prime the mature, infectious virion for entry into a new host cell (Flint et al, 2000). The pathway from initial infection to the completion of assembly lasts between 6 and 10 hours (Koch and Koch, 1985). By this point, infected cells have begun to break down as a result of abrogated protein synthesis, and virions usually exit through cell lysis, although in some cell types, poliovirus is shed nondestructively. Lysis can release as many as 100,000 new virions into the extracellular environment. This high yield is necessary; because only one percent of progeny virions go on to establish productive infections in other host cells.