Abstract
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.
Highlights
Chikungunya virus (CHIKV) re-emerged in 2004 and has caused unprecedented outbreaks in Asia and Africa since 2005
CHIKV nonstructural protein (nsP) will presumably together with host factors - assemble into replication and transcription complexes (RTCs) that associate with membrane structures derived from the plasma membrane and/or endosomes, as observed for other alphaviruses [5,6,7]
The complete genomes of the 13 CHIKV strains carrying the E1-A226V mutation (Table 1) that were available in GenBank at the time of in silico design (November 2009) were aligned using MAFFT [33] and the resulting consensus sequence formed the basis for the synthetic full-length cDNA clones
Summary
Chikungunya virus (CHIKV) re-emerged in 2004 and has caused unprecedented outbreaks in Asia and Africa since 2005. Assuming that CHIKV follows the typical alphavirus life cycle, proteolytic processing of the nonstructural polyproteins by the protease domain in nsP2 will lead to the release of nsP1, nsP2, nsP3, and nsP4. These nsPs and their precursors possess a variety of functions and the enzymatic activities, including protease, helicase, methyltransferase, and RNA-dependent RNA polymerase (RdRp) activity that drive CHIKV replication [5]. In addition to replication of its genomic RNA, CHIKV transcribes a subgenomic (sg) RNA encoding a precursor polyprotein that is processed by viral and cellular proteases into the structural proteins C, E3, E2, 6K and E1. CHIKV nsPs will presumably together with host factors - assemble into replication and transcription complexes (RTCs) that associate with membrane structures derived from the plasma membrane and/or endosomes, as observed for other alphaviruses [5,6,7]
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