NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly - PubMed (original) (raw)

NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly

Yinghong Ma et al. J Virol. 2008 Aug.

Abstract

A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

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Figures

FIG. 1.

FIG. 1.

(A) Organization of the chimeric HCV RNAs included in this study. Three genome-length HCV RNAs are shown, JFH1, HJ3, and HJ2, while Jtat2ANeo is a dicistronic subgenomic replicon containing nonstructural sequence derived from JFH1 virus. The JFH1 polyprotein coding sequence is shown as open boxes, and the H77 sequence is shaded. The base positions of the H77 and JFH1 nucleotides flanking the site of the fusion in the HJ2 chimera are shown, as is the location of the Q221L mutation within the helicase domain of NS3. (B) Virus yield assays showing the effect of the Q221L mutation on the efficiency of infectious virus assembly and release by various HCV RNAs. FT3-7 cells were transfected with HJ3, HJ2, and JFH1 RNAs with or without the Q221L mutation as indicated. At day 3 posttransfection, supernatant culture fluids were collected and assayed for released virus, while cell lysates were prepared by multiple freeze-thaw cycles and assayed for cell-associated virus. The data shown represent the means of the three replicate experiments, ± standard deviations. The titer (FFU/ml) of intracellular (shaded bars) and extracellular (open bars) virus was determined as described previously (51).

FIG. 2.

FIG. 2.

Replication of intergenotypic chimeric HJ3 (A) and JFH1 (B) RNAs with or without the NS3-Q221L mutation. Viral RNA abundance was measured by quantitative RT-PCR assay of cell lysates harvested at 4.5, 24, 48, 72, and 96 h after transfection of the indicated RNA into FT3-7 cells. The data represent a triplicate analysis, ± standard deviations. (C) Immunoblots showing HCV core protein abundance at 48, 72, and 96 h after transfection of the indicated RNAs; GAPDH was included as a loading control. (D) Subgenomic JFH1 replicon RNA replication assay. Relative SEAP activity induced by replication of Jtat2ANeo (lightly shaded bars) and Jtat2ANeo/QL (heavily shaded bars) at 24, 48, 72, and 96 h after transfection of the RNAs is shown. Results are normalized to the SEAP activity present at the same time point in media from cultures of cells transfected in parallel with the replication-defective Ntat2ANeo/ΔGDD RNA. The data shown are means ± ranges from two independent experiments.

FIG. 3.

FIG. 3.

Expression of core antigen at 2 and 4 days after transfection of cell cultures with the HJ3 and HJ3/QL RNAs. (A) Core antigen detected by indirect immunofluorescence microscopy. (B) The percentage of HCV RNA-transfected cells expressing HCV core antigen was quantified by FACS analysis. The length of the open doubleheaded arrow is proportional to the intensity of core staining and is greater in the graph depicting HJ3-transfected rather than HJ3/QL-transfected cells.

FIG. 4.

FIG. 4.

Laser-scanning confocal microscopic imaging. FT3-7 cells were transfected with JFH1, HJ3, or HJ3/QL RNA and 2 days later washed and fixed, as described in Materials and Methods, and labeled with probes specific for core protein (green), NS5A (red), and neutral lipids (white) (A), or core protein (green), NS3 (red), and neutral lipids (white) (B), prior to examination in a Zeiss LSM 510 Meta instrument. At the right is an enlarged area from the merged image showing a single lipid droplet (see box).

FIG. 5.

FIG. 5.

Equilibrium gradient ultracentrifugation of viral particles present in cell culture supernatant and cell lysates collected 3 days after transfection of FT3-7 cells with RNA encoding HJ3 (left panels) and HJ3/QL (right panels). (A) Distribution of infectious virus present in gradient fractions as determined by FFU assay. Only HJ3/QL produced infectious virus, and this was present in both lysates and supernatant fluids. HJ3 did not produce detectable quantities of infectious virus (<10 FFU/ml). (B) Core antigen as measured by quantitative ELISA in each fraction. (C) Semiquantitative RT-PCR assay of HCV RNA in fractions from gradients loaded with cell culture supernatant fluids (top panels) or cell lysates (bottom panels). The open and closed arrows indicate two distinct slowly sedimenting (open) and rapidly sedimenting (closed) species of HCV RNA-containing particles present in supernatants.

FIG. 6.

FIG. 6.

Rate zonal centrifugation of cell lysates derived from FT3-7 cells transfected with the HJ3, HJ3/QL, or JFH1/QL RNAs or Huh7/1-191 cells which conditionally express only the HCV core protein (23). Cell lysates were prepared by multiple rounds of freeze-thawing and centrifuged in preformed sucrose density gradients. The sedimentation profiles of infectious virus present in lysates of HJ3/QL- and JFH1/QL-transfected cells are shown at the top. No infectious virus was present in lysates from the HJ3-transfected cells. The immunoblots of core protein present in the top 20 fractions of the gradients are shown at the bottom. No detectable core protein was present in fractions beyond fraction 20 (data not shown). Fractions prepared from the four gradients depicted had density profiles indistinguishable from that shown.

FIG. 7.

FIG. 7.

Lack of effect of the Q221L mutation on NS3-associated enzyme activities. (A) SDS-PAGE separation of purified GST-fused JFH1 NS3 proteins with or without the Q221L mutation. *, a truncated NS3 protein that was reactive with anti-NS3 antibody. (B) Helicase (unwindase) activities were measured with a radiolabeled double-stranded oligonucleotide substrate using a range of concentrations of the indicated GST-NS3 proteins or GST alone. (C) NTPase activities of the GST-NS3 proteins were measured by determining release of free phosphate. (D) The protease activity of GST-NS3 proteins (or GST alone) was assessed in the presence or absence of added NS4A cofactor peptide in a FRET assay. The velocity of the reaction (_V_max) was calculated for a range of GST-NS3 protein concentrations from measurements of the product of the reaction taken at 5-min intervals over a 30-min period. (E) In vitro _cis_-processing of 35S-labeled JFH1 polyprotein segments representing NS2-NS3-NS4A and containing either the wild-type Gln-221 residue or the mutant Leu-221 residue. Samples were collected from a cell-free translation reaction carried out in rabbit reticulocyte lysates at 15, 30, 60, and 180 min and subjected to SDS-PAGE analysis followed by autoradiography.

FIG. 8.

FIG. 8.

(A) Impact of substitutions within the NS3 221 codon on release of infectious virus by cells transfected with HJ3 RNA or various mutants derived from it. The data shown represent the means plus standard deviations of virus released 2 days after transfection of the indicated RNAs in replicate experiments (uppercase letters refer to the amino acid present at the NS3-221 position, while lowercase letters in parentheses indicate the nucleotide sequence of the NS3 codon 221 in each). The defect in virus production that is evident in the HJ3 chimera containing NS3-Q221 was rescued by introduction of a Q221L mutation (irrespective of the nucleotide sequence of codon 221) or to a lesser extent by the Q221I and Q221N mutations. Although there was no detectable virus produced by an RNA encoding Val-221, continued passage of transfected cells gave rise to a mutated virus encoding Leu-221, as shown by an arrow in the lower part of the figure. Virus yield from other mutants, and second-site mutations arising from continued passage of transfected cells, are as shown. *Leu-221 indicates that in addition to the mutation of Val-221 to Leu-221, the Val-221 mutant also acquired a Leu-to-Ser substitution at residue 66 of the NS2 sequence (L875S in the H77 polyprotein). The V286 and V399+V286 mutants encode the wild-type Gln-221 residue. Infectious virus was measured in supernatant fluids 2 days after RNA electroporation. The detection limit was 10 FFU/ml. (B) Core protein expression as determined by immunofluorescence 2 and 4 days after transfection of cells with the indicated RNAs.

FIG. 9.

FIG. 9.

Location of mutations promoting assembly of HJ3 virus within the crystallographic structure of the NS3 helicase from the genotype 1b BK strain of HCV (PDB 1CU1) (48). (A) Two views of the major NS3 helicase subdomains, showing the side chains in space-filling view for residues Gln-221 (red), Ile-286 (orange), and Val-399 (yellow). Gln-221 and Ile-286 are conserved between the JFH1 and BK virus NS3 molecules, while residue 399 is Ile in JFH1 and Val in the BK virus. The protein backbone of subdomain 1 (NTPase) is displayed in blue, with the Walker A and Walker B motifs highlighted in green, while the backbone of subdomain 2 (RNA binding activity) is displayed in brown. (B) Expanded view of subdomain 1, showing the relationship of the side chains of Ile-286 (orange, space-filling view), Gln-221 (red, stick-and-ball view), and the flanking tyrosines Tyr-223 and Tyr-118 (green and yellow, respectively; stick-and-ball view).

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