Next, ATP along with 32P–ATP were added and reactions were terminated after 30?min by the addition of EDTA and formamide. for therapeutic development. Introduction Mosquito-borne Zika virus (ZIKV) is a member of the Flaviviridae family first identified in 1947 in the Zika Forest region in Uganda1C3. Although ZIKV infections have historically caused moderate symptoms and can infrequently lead to Guillain-Barr syndrome, the recent massive spread of the virus to the Americas has caused the World Health Organization to declare an international health emergency1C3. This is due to the ability of the virus to cause a rare neurological condition in infants called microcephaly which is usually characterized by a significantly reduced head size and impaired neurological and motor skill development4. Strikingly, ZIKV is not only transmitted from mother to fetus, but also via sexual intercourse1. Recent evidence in mice also indicates that ZIKV contamination in males CBR 5884 may cause sterility5. Hence, our current knowledge of the detrimental effects of ZIKV contamination appears to be limited. ZIKV has spread to 60 countries including the United States where ZIKV infections have been reported in Florida and in Texas6,7. Although endeavors are underway to develop a vaccine, this can take several years and is not necessarily guaranteed to be effective. Thus, an alternative strategy to combat ZIKV, such as the development of anti-viral drug inhibitors, is an important area of research. Based on the past successes of using polymerase inhibitors to treat human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, drug inhibitors of the ZIKV RNA-dependent RNA polymerase (RdRp) are CBR 5884 likely to be similarly effective in treating ZIKV infections8,9. However, our current knowledge of how the ZIKV replication machinery CBR 5884 functions is limited. All known Flavivirus RdRp enzymes are directly fused to a methytransferase (MTase) through a flexible linker within the non-structural 5 (NS5) protein that is essential for replication (Fig.?1a). The MTase performs 5 RNA capping by facilitating guanine-N7 and nucleoside-2-O methylation actions10C12. RNA capping promotes RNA stability, efficient translation and evasion of the host immune response11,13. Yet, why the MTase is usually directly fused to RdRp in the Flavivirus genus remains unclear. A plausible explanation is that these linked enzymes cooperate during RNA synthesis. Studies of related Dengue virus (DENV) report conflicting findings on the effects of the MTase on RdRp RNA synthesis activity. For example, stimulation and suppression of DENV RdRp by the MTase have been reported14C16, whereas another study showed that MTase had no effect on DENV RdRp activity17. Intriguingly, mutation of key residues involved in DENV MTase-RdRp interactions primarily resulted in an increase in RdRp initiation and elongation activities study using ZIKV proteins suggests that the MTase only significantly contributes to RdRp RNA elongation activity, which contrasts previous reports on DENV14. Clearly, detailed biochemical studies are required to unequivocally determine whether the effects of MTase on RdRp activity are universal among Flaviviruses or vary CBR 5884 between different members such as ZIKV. Open in a CBR 5884 separate window Physique 1 MTase CR6 is essential for RdRp elongation and initiation. (a) Schematic of ZIKV NS5 protein. (b) Comparison of ZIKV (right; PDB 5tfr) and DENV (left; PDB 4v0q) NS5 crystal structures highlighting different RdRp orientations. F motif, fingers, palm and thumb subdomains are indicated in ZIKV NS5 (right). (c) Denaturing SDS gels showing purified ZIKV NS5 (left) and RdRp (right). (d) Schematic of elongation assay (top). Denaturing gels showing elongation by RdRp and NS5 on indicated template (left). Bar chart showing the relative intensities of RdRp and NS5 elongation products generated after 30?min. value decided.
Next, ATP along with 32P–ATP were added and reactions were terminated after 30?min by the addition of EDTA and formamide