Construction and mutagenesis of an artificial bicistronic tick-borne encephalitis virus genome reveals an essential function of the second transmembrane region of protein e in flavivirus assembly

Abstract

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3' noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.

FIG. 1.

Bicistronic mutants derived from TBEV. (A) Schematic drawing of the wt and the bc TBEV genome (not drawn to scale). For the construction of TBEV-bc, the prM and E coding regions were deleted from their natural position within the long open reading frame and reinserted together with an heterologous EMCV-IRES element in place of the variable region of the 3′-NCR as indicated in the drawing. The transmembrane regions present in the structural proteins are indicated by black bars. Filled triangles indicate copies of the second transmembrane region of protein E (E-TM2), which in the wt genome simultaneously serves as a signal sequence for protein NS1. In the bc mutant, TM2 is present in two copies, once as a signal sequence for NS1 and once as part of the protein E anchor. To prevent genetic instability, the sequence of the second E-TM2 copy was wobbled, and the nucleotide and amino acid sequences of this altered element are shown below with the altered (wobbled) nucleotides of the synonymous codons underlined. (B) The three functional domains of E-TM2 (n, polar region, h, central hydrophobic region, c, domain required for signalase cleavage) are depicted. The amino acid sequence changes in the three truncation mutants and mutant bc YF, in which E-TM2 was replaced by the corresponding region from yellow fever virus, are shown below. Asterisks depict the residues in the YFV sequence that are different from those in the TBEV sequence.

FIG. 2.

Protein expression and RNA replication of TBEV-bc in BHK-21 cells. (A and B) Expression of protein E (A) or NS1 (B) was determined by immunostaining with specific monoclonal antibodies and FACS analysis from 1 to 5 days after transfection of BHK-21 cells with bc or wild-type RNA. The mean fluorescence intensity of the brightest 10% of each cell population, after subtracting the background value obtained for mock-transfected control cells (Mock), is plotted in arbitrary units (a.u.) on a logarithmic scale. Immunofluorescence pictures of cells 4 days after transfection are shown to the right. Intracellular RNA levels per cell for the bc mutant and its parental replicons ΔME and ΔME-EGFP, as well for as a replication-negative control (ΔNS5), were determined at the indicated time points by quantitative PCR. RNA detected 2 h posttransfection corresponds to residual input RNA.

FIG. 3.

RNA export kinetics of the bc mutant. (A) The concentration of viral RNA in the supernatants of BHK-21 cells transfected with bc RNA (open squares) or monocistronic ΔR88 RNA (filled triangles) was monitored between 2 h and 4 days posttransfection. Values obtained for mock-transfected cells (Mock) were below the cutoff value 10² (dashed line) at all times. (B) The percentage of the total RNA (intra- plus extracellular RNA, both measured by real-time PCR) released from the cells was calculated at each time point using the same RNA samples whose results are shown in panel A as well as a control replicon (ΔCME) from which all of the structural protein genes had been deleted and which was therefore unable to make virus particles (open circles). Mean values from two independent experiments (each consisting of double values) with error bars indicating standard deviations are shown. Values obtained 2 h posttransfection (marked by an asterisk) represent residual input RNA (removed by a subsequent medium change) rather than exported RNA.

FIG. 4.

Sedimentation analysis of particles produced by the bc mutant (open squares) or wt virus (filled triangles). Particles harvested from supernatants of RNA-transfected cells and concentrated by ultracentrifugation were fractionated by rate zonal centrifugation on continuous 12 to 50% sucrose gradients. The amount of viral RNA in each 0.6-ml fraction was determined by quantitative PCR and is plotted as a percentage of the total RNA.

FIG. 5.

Infectivity of bc mutants in cell culture. BHK-21 cells were transfected with wt or bc RNA (or mock transfected) (A) and with various bc mutants (B) as indicated on the left. Supernatants were transferred to fresh cells 6 days later, and this passaging procedure was continued for a second round or several more cycles as indicated by arrows. Infection of cells was detected 3 days after transfection or infection by IF staining using a polyclonal serum that recognizes both structural and nonstructural proteins of TBEV. Results are shown for transfected cells and passages 1 and 2 as indicated at the top.

FIG. 6.

Neurovirulence of wt virus and mutant bc upon intracranial inoculation of suckling mice. Supernatants of transfected cells were diluted to the same virus concentration (2,000 RNA molecules/μl). Survival of mice was recorded over a 28-day period.

FIG. 7.

Comparison of RNA export capacity of the different bicistronic mutants. Intracellular and extracellular RNA was quantified by real-time PCR 72 h after transfection of BHK-21 cells with RNA, and the calculated percentage of the total RNA in the cell supernatant is shown for each mutant. Values represent means from the results of two independent experiments (each consisting of double values) with error bars indicating standard deviations.