Structure and function analysis of the essential 3′X domain of hepatitis C virus

Abstract

The 3 ′ ′ ′ ′ ′ X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3 ′ ′ ′ ′ ′ X mutants that favor one of the two previously proposed conformations comprising either two or three stem-loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3 ′ ′ ′ ′ ′ X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3 ′ ′ ′ ′ ′ X domain does not occur as a result of a transition between two-and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3 ′ ′ ′ ′ ′ X homodimers.

The low-resolution structural models of hepatitis C virus RNA subdomain 5BSL3.2 and its distal complex with domain 3′X point to conserved regulatory mechanisms within the Flaviviridae family

“…However, these models gave rise to poorer fitting with the experimental SAXS curve (χ 2 = 26.7 relative to 7.91), less overlap with the dispersion envelopes and significantly poorer inter-domain stacking ( Supplementary Figure S10 ). This result confirmed previous studies based on NMR spectroscopy, gel electrophoresis, thermal melting and functional experiments ( 10 , 11 , 20 ), indicating that domain 3′X does not require a conformational transition to a SL1, SL2 and SL3 hairpin structure to interact with 5BLS3.2 or regulate the virus replication cycle.…”

“…This helix is coaxially stacked with the upper stem of 5BSL3.2 and the lower stem of subdomain SL2′. Although this structure was obtained with a dimerization-defective 3′X domain (Figure 1 ), previous NMR evidence indicated that the wild-type sequence is capable of forming a similar structure (( 11 , 20 ) and Supplementary Figure S9B ). As observed for 5BSL3.2 and 3′X, the SAXS data revealed that the complex adopted a well-defined structure with limited flexibility (Figure 2H and Supplementary Figure S6C ).…”

“…Previous NMR analyses of wild-type and dimerization-defective 3′X sequences in contact with the 5BSL3.2 cre26 hairpin indicated that within the 3′X domain, subdomain SL1′ and the lower double-helical stem of subdomain SL2′ remained undisturbed upon complex formation ( 10 , 11 , 20 ). The changes only affected the upper region of subdomain SL2′ where the k nt are base-paired, and involved the establishment of new Watson-Crick pairs between these nt and the k ′ nt of the 5BSL3.2 apical loop ( 10 , 11 ). On the other hand, chemical shift perturbations in cre26 were mostly restricted to the apical loop ( 10 ).…”

“…This 3′X k sequence is part of a self-complementary 16-nt segment termed dimer linkage sequence (DLS) that has been shown to promote the dimerization of virus genomes in vitro ( 17–21 ) (Figure 1 ). The 5SL3.2–3′X k ′- k contact has been reported to be essential for virus replication ( 7 , 8 ) and translation ( 11 , 14 , 22 ). Not surprisingly, all of the components of this RNA–RNA interaction network, subdomain 5BSL3.2, IRES subdomain IIId, SL9005 and domain 3′X, are evolutionarily conserved within the extremely variable genome of HCV.…”

Hepatitis C Viral Replication Complex

“…The X-tail contains three SL structures [ 82 ]. All of these three SLs are essential for RNA replication [ 83 ] and barely tolerate any mutations [ 84 , 85 , 86 ]. X-tail is probably the main regulatory element for the initiation of negative-strand synthesis.…”