An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

Ouabain-sensitive K(+)-ATPase in epithelial cells from guinea pig distal colon

We have previously shown that an ouabain-sensitive H(+)-K+ exchange mechanism may be present in the apical membrane of guinea pig distal colon [Y. Suzuki and K. Kaneko. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G979-G988, 1989]. The present study is aimed to demonstrate the presence of an ATPase responsible for this exchange. ATPase activity was determined in the crude membrane fraction of the colonic epithelial cell homogenate. ATPase activity under Na(+)-free conditions was increased by the addition of K+, with a half-maximal effect at 55 microM. This increase was completely abolished by 1 mM ouabain, suggesting the presence of an ouabain-sensitive K(+)-ATPase. The ouabain-sensitive K(+)-ATPase activity was inhibited by vanadate (100 microM) and N,N'-dicyclohexylcarbodiimide (100 microM) but was resistant to oligomycin (4.5 micrograms/ml) and NaN3 (1 mM). The ouabain-sensitive K(+)-ATPase activity was observed in the distal but not in the proximal colon, whereas Na(+)-K(+)-ATPase activity was distributed along the entire colon. Omeprazole (40 microM) reduced the colonic K(+)-ATPase activity by 31 +/- 6%, whereas it reduced the gastric K(+)-ATPase activity by 78 +/- 8%. These results suggest that the ouabain-sensitive K(+)-ATPase as demonstrated here is responsible for the colonic H(+)-K+ exchange. This ATPase could be similar to but is not identical with either Na(+)-K(+)-ATPase or gastric H(+)-K(+)-ATPase.