Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function

Abstract

Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied in trans and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in trans, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.

FIG. 1

Expression of DEN NS1 and NS1-2A. BHK-21 cells (mock infected or infected with DEN-2 [MOI, 10] for 22 h) or BHK-SINrep21 derivatives were metabolically labeled for 3 h. NS1 was immunoprecipitated from equivalent portions of cellular SDS extracts or conditioned labeling media and eluted into loading buffer. Half of the eluate was boiled prior to electrophoresis. Numbers to the left indicate relative molecular masses in kilodaltons.

FIG. 2

DEN NS1 does not complement YFΔSK. (A) Growth of YFΔSK on cells expressing DEN NS1 (■) or YF NS1 (⧫). Cells were infected with YFΔSK (MOI, 10) for 1 h. The inoculum was removed, the cells were briefly rinsed with PBS, and fresh growth medium was added. Virus growth media were sampled at 0, 12.5, 23, 37, and 46 h postinfection, clarified, and stored at −80°C. Virus titers were determined on BHK-SINrep21/YF NS1 cells and plotted here on a semilogarithmic scale. (B) RPA of viral minus strands. RNAs were harvested at 0, 4, 8, or 20 h postinfection. Equivalent portions of protected minus-strand reactions were subjected to denaturing electrophoresis, dried, and exposed to film. Numbers above lanes 2 to 5 refer to the numbers of synthetic minus-strand molecules analyzed in parallel; numbers above lanes 6 to 19 refer to hours postinfection. The 10⁹ standard, which appeared as a smear in this exposure, did yield a distinct band in a lighter exposure and is included here to further demonstrate the sensitivity of this assay to the quantity of input RNA.

FIG. 3

Selection of YFΔSKden by RNA transfection. BHK-SINrep21 cells expressing YF NS1, DEN NS1, or GFP were transfected with YFΔSK RNA and plated on 100-mm dishes. Following incubation, the cells were fixed and stained for plaque formation.

FIG. 4

Growth of YFΔSK on various cell populations. (A) BHK-SINrep21 cell monolayers (expressing YF NS1 [●], DEN NS1 [□], or GFP [⧫]) were infected with YFΔSK (MOI, 10) for 1 h, followed by three washes with PBS. At 12-h intervals, the growth media were removed and replaced with fresh media, clarified, and stored at −80°C. Virus titers were determined on YF NS1-expressing cells and plotted on a semilogarithmic scale. (B) The titers of the same samples from panel A determined on DEN NS1-expressing cells. The broken line indicates the detection limit of these plaque assays. The data represent the cumulative virus yields and are expressed as the means of the sum ± standard deviations for three independent experiments.

FIG. 5

YFΔSKden can utilize DEN NS1 for RNA replication. (A) Growth analysis of independently derived YFΔSKden 1.7, 2.5, and 3.1. BHK-SINrep21 cells expressing YF NS1 (solid symbols) or DEN NS1 (open symbols) were infected with the indicated YFΔSKden isolates (MOI, 10) for 1 h, followed by three washes with PBS. Virus growth media were sampled at 0, 24, and 48 h postinfection, clarified, and stored at −80°C. Virus titers were determined on BHK-SINrep21/YF NS1 cells. The lower limit of the graph indicates the limit of detection in the plaque assays. (B) RNA replication of YFΔSKden. BHK-SINrep21 cells expressing YF NS1 (lanes 2 to 7), DEN NS1 (lanes 8 to 13), or GFP (lanes 14 to 19) were infected with the indicated viruses (MOI, 10). Total cellular RNAs were harvested at 16 h postinfection and subjected to RPA analysis of viral plus strands. MOCK, mock infection.

FIG. 6

Summary of YFΔSKden sequence analysis. Shown at the top is the YFΔSK genome. Thick bars below the genome indicate regions of the viral cDNAs that were sequenced. A single point substitution at position 5783, coding for a change of Asn (N) to Tyr (Y), is indicated.

FIG. 7

NS4A-N42Y can confer the YFΔSKden phenotype. The specific infectivities of wild-type YFΔSK RNA (white bars) or mutant YFΔSK RNA (gray bars) were assayed by transfection of cells expressing YF NS1 (left bars) or DEN NS1 (right bars). Values represent the log geometric means ± standard deviations for four independent experiments.

FIG. 8

Model of NS4A topology and secondary structure. (A) The topology of NS4A/2K was modeled as described in Materials and Methods. The arrows, open box, and black dot indicate viral serine protease cleavage sites, a signal peptidase cleavage site, and the approximate location of NS4A residue 42, respectively. (B) Several flavivirus NS4A coding regions were aligned with YF NS4A residues 11 to 60. The virus sequences shown (and their GenBank accession numbers) are as follows: YF 17D (P03314), DEN-1 (P33478), DEN-2 (P123823), DEN-3 (P27915), DEN-4 (P09866), Japanese encephalitis (JE) (P32886), Kunjin (KUN) (P14335), tick-borne encephalitis (TBE) (P14336), and West Nile (WN) (P06935). Shown below the alignment is a prediction of the secondary structure: H, helix; L, loop; E, strand; dot, not predictable. Also shown is the beginning of the first putative transmembrane domain, depicted as a box. The location of YF NS4A residue 42 is indicated above the alignment by a black dot.