Single‐molecule imaging reveals the translocation and DNA looping dynamics of hepatitis C virus NS3 helicase

Abstract

Non-structural protein 3 (NS3) is an essential enzyme and a therapeutic target of hepatitis C virus (HCV). Compared to NS3-catalyzed nucleic acids unwinding, its translation on single stranded nucleic acids have received relatively little attention. To investigate the NS3h translocation with single-stranded nucleic acids substrates directly, we have applied a hybrid platform of single-molecule fluorescence detection combined with optical trapping. With the aid of mechanical manipulation and fluorescence localization, we probed the translocase activity of NS3h on laterally stretched, kilobase-size single-stranded DNA and RNA. We observed that the translocation rate of NS3h on ssDNA at a rate of 24.4 nucleotides per second, and NS3h translocates about three time faster on ssRNA, 74 nucleotides per second. The translocation speed was minimally affected by the applied force. A subpopulation of NS3h underwent a novel translocation mode on ssDNA where the stretched DNA shortened gradually and then recovers its original length abruptly before repeating the cycle repetitively. The speed of this mode of translocation was reduced with increasing force. With corroborating data from single-molecule fluorescence resonance energy transfer (smFRET) experiments, we proposed that NS3h can cause repetitive looping of DNA. The smFRET dwell time analysis showed similar translocation time between sole translocation mode versus repetitive looping mode, suggesting that the motor domain exhibits indistinguishable enzymatic activities between the two translocation modes. We propose a potential secondary nucleic acids binding site at NS3h which might function as an anchor point for translocation-coupled looping.

Branched unwinding mechanism of the Pif1 family of DNA helicases

“…Repetitive unwinding/translocation activity on DNA, monitored by FRET and/or protein-induced fluorescence enhancement changes, has been reported for multiple helicases/translocases (1,3,4,7,8,10,13,14,49), including Pif1 (6,12,43,47). The repetitive cycling behavior has been shown to be ATP dependent, as expected for a phenomenon driven by a motor protein; however, how ATP affects the multiple steps within the cycle has not been extensively characterized.…”

“…The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity. single molecule | DNA unwinding | helicase | DNA replication G rowing experimental evidence indicates that multiple helicases/translocases display a peculiar behavior in which a single motor protein molecule can repetitively translocate on nucleic acid (1)(2)(3)(4)(5)(6) or unwind double-stranded DNA (7)(8)(9)(10)(11) and G-quadruplex structures (6,(12)(13)(14). How this repetitive enzymatic activity is used for function remains unclear, but it may be linked to the specific pathway in which these helicases operate, the nature of the substrate itself, and the directionality of unwinding.…”

Approaches for measuring the dynamics of RNA–protein interactions

“…Optical traps thus provide information that is beyond the scope of other methods. An optical trap has been combined with single molecule fluorescence detection to directly measure coordination of multiple processes in protein-DNA systems (Lin et al, 2017;Lin & Ha, 2017b), but to our knowledge, not yet in an RNA-protein system.…”

In vitro methods for testing antiviral drugs

“…Virus-encoded ATPase-driven helicases have been identified in numerous human pathogens. Helicases from several (+)RNA viruses have been characterized, including NS3 helicases from flaviviruses such as dengue virus, HCV, West Nile virus, yellow fever virus, and Japanese encephalitis virus (Cao et al, 2016;Gu and Rice, 2016;Jain et al, 2016;Lin et al, 2017;Nedjadi et al, 2015;Wu et al, 2005). SARS and MERS coronaviruses encode nsp13 with helicase activity (Adedeji and Lazarus, 2016;Hao et al, 2017;Seybert et al, 2000).…”