Genetic dissection of interaction between poliovirus 3D polymerase and viral protein 3AB

Abstract

Poliovirus RNA-dependent RNA polymerase 3D and viral protein 3AB are both thought to be required for the initiation of RNA synthesis. These two proteins physically associate with each other and with viral RNA replication complexes found on virus-induced membranes in infected cells. An understanding of the interface between 3D and 3AB would provide a first step in visualizing the architecture of the multiprotein complex that is assembled during poliovirus infection to replicate and package the viral RNA genome. The identification of mutations in 3D that diminish 3D-3AB interactions without affecting other functions of 3D polymerase is needed to study the function of the 3D-3AB interaction in infected cells. We describe the use of the yeast two-hybrid system to isolate and characterize mutations in 3D polymerase that cause it to interact less efficiently with 3AB than wild-type polymerase. One mutation, a substitution of leucine for valine at position 391 (V391L), resulted in a 3AB-specific interaction defect in the two-hybrid system, causing a reduction in the interaction of 3D polymerase with 3AB but not with another viral protein or a host protein tested. In vitro, purified 3D-V391L polymerase bound to membrane-associated 3AB with reduced affinity. Poliovirus that contained the 3D-V391L mutation was temperature sensitive, displaying a pronounced conditional defect in RNA synthesis. We conclude that interaction between 3AB and 3D or 3D-containing polypeptides plays a role in RNA synthesis during poliovirus infection.

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

“…The V391L mutation (Fig. 5A) was originally identified in a two-hybrid screen for polymerase mutations that interfered with the binding of 3D polymerase to 3AB, its membrane tether (Hope et al 1997;Lyle et al 2002b). The temperature sensitivity of the V391L mutant virus is consistent with the hypothesis that, at the nonpermissive temperature, the defective 3D-3AB interaction allows the mutant polymerase to be released from its membrane tether, thus reducing its effective concentration in the RNA replication complex.…”

“…HeLa cells were seeded at a density of 7.5 3 10 5 cells per 6-cm plate 24 h prior to infection. Cells were infected with wild-type or V391L mutant poliovirus as described (Hope et al 1997) at a multiplicity of infection of 20 plaque-forming units/cell. For RNA accumulation assays, after 30 min adsorbtion at 37°C, cells were incubated at 32.5°C or 39.5°C for the indicated times.…”

“…Interactions between 3C and 3D have been observed by glutaraldehyde cross-linking and enzymatic function ; two different potential 3C-3D interaction surfaces have been observed crystallographically (Marcotte et al 2007). Oligomerization of purified 3D RNA-dependent RNA polymerase in solution has been demonstrated by turbidity assays, electron microscopy (Lyle et al 2002a), glutaraldehyde cross-linking , and two-hybrid experiments (Hope et al 1997;Xiang et al 1998). The functional nature of such polymerase-polymerase interactions has been supported by observed hallmarks of oligomeric activity such as intermolecular enzymatic uridylylation (Richards et al 2006), cooperative RNA binding (Pata et al 1995;Beckman and Kirkegaard 1998), cooperative RNA elongation (Pata et al 1995;Hobson et al 2001), defective phenotypes of mutant viruses with mutations at protein-protein interfaces (Hobson et al 2001;Pathak et al 2002), and dominant phenotypes of several polymerase alleles (Crowder and Kirkegaard 2005).…”

The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity

“…15 Protein 3AB also associates with itself, 3CD pro , 3D pol and proteins from the P2 genome region. [16][17][18][19][20][21][22] Although 3AB normally associates non-specifically with RNA, 22,23 binding to 3CD produces a complex that associates strongly with the 5' cloverleaf structure of the viral genome ( Fig. 1) in vitro and may be involved with anchoring the genome to the vesicles.…”

Strategies to develop antivirals against enterovirus 71

“…By analyzing the enviroxime-resistant mutants, the target site of enviroxime was identified on viral protein 3A [52]. The viral protein 3A and its precursor 3AB play the key roles in formation of enterovirus replication complex [53,54]. Development of anti-vrials targeting on 3A or 3AB may be a successful strategy for inhibiting EV71 replication.…”