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Dr. Rudy Beran Post-doc
rudolf.beran@yale.edu
RNA
substrate tracking by the NS3 helicase of Hepatitis C virus
The NS3 helicase is a DExH/D family protein from Hepatitis C virus (HCV)
that has the ability to re-arrange nucleic acid secondary structure in an
ATP-dependent manner. In addition, this protein is essential for HCV replication.
The NS3 helicase may have to re-arrange long stretches (i.e. several kilobases)
of genomic RNA during replication in order to permit copying by NS5B, the
HCV RNA polymerase. Curiously, the NS3 helicase has been demonstrated to
have poor processivity on RNA duplexes relative to other characterized DExH/D
RNA helicases such as the NPH-II helicase of vaccinia virus. Given this
situation, it is of interest to use modified substrates to determine how
NS3 recognizes and moves along an RNA substrate in comparison to other RNA
helicases. The manner in which a helicase tracks along a duplex can be elucidated
by designing nucleic-acid duplexes with perturbations such as nicks, polyglycol
linkers, or riboabasic residues. Previous work in our lab has shown that
the NPH-II helicase requires continuous contact with the ribose-phosphate
backbone of the loading strand of an RNA duplex in order to unwind.
We have determined that the NS3 helicase does not track along an RNA duplex
in the same manner as the NPH-II helicase. We have observed that the NS3
helicase is similar to the NPH-II helicase in that it can not tolerate a
loading strand that lacks covalent continuity. However, the NS3 helicase
can unwind RNA duplexes that lack nucleic acid continuity due to the presence
of polyglycol linkers on the loading strand. Thus, it appears that the NS3
helicase does not require specific contacts with the ribose-phosphate backbone
in order to track along an RNA duplex. Moreover, these results suggest that
the NS3 helicase differs from the NPH-II helicase in that it can step over
perturbations on the loading strand. In the future, we plan to examine how
the presence of other HCV proteins (i.e. the NS5B polymerase) that are known
to physically interact with NS3 affect its unwinding characteristics.
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