Alphavirus RNA synthesis and non-structural protein functions

Abstract

The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

Fig. 1.. Alphavirus RNA synthesis scheme. After infection the alphaviral genomic RNA is translated to yield the P1234 polyprotein, which following proteolytic cleavage via nsP2 forms the minus-strand replicase complex consisting of P123 and nsP4. Together, P123 and nsP4 initiate the synthesis of the minus-strand RNA, which serves as a template for synthesis of the genomic and subgenomic RNAs. Further processing of the P123 polyprotein, to nsP1 and P23, results in a switch from minus-strand synthesis to positive-strand synthesis, resulting in the production of genomic and subgenomic RNAs. The P23 polyprotein is exceptionally short-lived, with a presumed half-life of a few seconds. Proteolytic cleavage of P23 into nsP2 and nsP3 individually results in the fully cleaved viral replicase complex which, whilst capable of genomic and subgenomic RNA synthesis, strongly favours production of the subgenomic RNA.

Fig. 2.. Alphavirus non-structural proteins. Shown are domain organization of the individual alphaviral non-structural proteins nsP1, nsP2, nsP3 and nsP4. Recognized domains, in terms of either structural or genetic evidence, are indicated as grey boxes in their relative position in their respective proteins; specific features, as indicated on their respective proteins, are briefly listed below. nsP1: H designates the histidine residue determined to bind covalently to the ^7MeGMP moiety; MB1 and MB2 indicate the sites of the membrane-binding amphipathic helix and the palmitoylation site of nsP1, respectively. MTase, methyltransferase; GTase, guanylyltransferase. nsP2: NLS1 and NLS2 indicate the sites of canonical nuclear localization sites; the cysteine and histidine residues of the protease active site are indicated with a C and H, respectively. NTPase, nucleoside triphosphatase. nsP3: the location of the zinc ion coordination site is denoted with Zn; the presence of ampiphysin interaction sites is denoted with Ampi. AUD, alphavirus unique domain. nsP4: the functional GDD catalytic triad of RdRp is shown.

Fig. 3.. Alphavirus conserved RNA sequence elements (CSEs): schematic of the alphaviral genomic RNA with conserved RNA elements indicated in their relative positions on the genomic RNA. The nature of the conserved elements is colour coded to indicate whether RNA secondary structure, primary nucleotide sequence or both is conserved. Exceptions to either element location or presence are indicated. RRV, Ross river virus; RSE, repeat sequence element; URE, U-rich element.