Structural and functional studies of alphavirus replication proteins, nsP1 and nsP4

Shailly Tomar, Purdue University

Abstract

Alphaviruses encode four replication proteins (nsP1-nsP4) that are an integral part of the viral RNA replication complex. nsP4 is the RNA-dependent RNA polymerase (RdRp) that is essential for viral RNA synthesis. To pursue structural and functional studies of the RdRp, Sindbis virus (SINV) nsP4 was produced in E. coli. Due to degradation problems, expression of the full-length protein was unsuccessful. However, the core catalytic domain of nsP4 (Δ97nsP4, a deletion of the N-terminal 97 amino acids) was stably expressed and purified to homogeneity. We have identified that purified Δ97nsP4 possesses terminal adenylyl transferase (TATase) activity, because it catalyzed the specific addition of adenine to the 3' end of acceptor RNA in the presence of divalent cations. Furthermore, Δ97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP and dATP) to the acceptor RNA in the absence or presence of all other nucleotides. Δ97nsP4 possessing a GDD to GAA mutation inactivates the TATase activity. However, a GDD to SNN mutation did not inactivate the enzyme but reduced its activity to ∼45% of wild-type in the presence of Mg+2. Furthermore, the GDD to SNN mutation had no effect on the TATase activity in the presence of Mn+2 ions. Identification of Δ97nsP4 TATase activity suggests a novel function of nsP4 in polyadenylation of the alphavirus genome. A high throughput experiment was also performed for cloning and expression of nsP4 from various alphaviruses. The core catalytic domain of nsP4 from Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV) were expressed and purified successfully. nsP1, another alphavirus replication protein, is the capping enzyme containing S-adenosyl-L-methionine-dependent methyltransferase (MTase) and divalent cation-dependent guanylyltransferase (GTase) activities. SINV nsP1 was expressed in E. coli and purified. We demonstrated that the recombinant SINV nsP1 was enzymatically active by performing nsP1 MTase and GTase assays. Purified replication proteins (nsP1 and nsP4) were used for crystallization trials, but crystals were not observed perhaps due to the conformational heterogeneity of the samples. However, some needle-like crystals were produced from SINV nsP4 (98-599 residues) and further optimization of the crystallization condition may produce good quality protein crystals for structural studies.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Biochemistry|Virology

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