The prM-independent packaging of pseudotyped Japanese encephalitis virus
© Lee et al. 2009
Received: 20 April 2009
Accepted: 30 July 2009
Published: 30 July 2009
As noted in other flaviviruses, the envelope (E) protein of Japanese encephalitis virus (JEV) interacts with a cellular receptor and mediates membrane fusion to allow viral entry into target cells, thus eliciting neutralizing antibody response. The formation of the flavivirus prM/E complex is followed by the cleavage of precursor membrane (prM) and membrane (M) protein by a cellular signalase. To test the effect of prM in JEV biology, we constucted JEV-MuLV pseudotyped viruses that express the prM/E protein or E only. The infectivity and titers of JEV pseudotyped viruses were examined in several cell lines. We also analyzed the neutralizing capacities with anti-JEV sera from JEV-immunized mice. Even though prM is crucial for multiple stages of JEV biology, the JEV-pseudotyped viruses produced with prM/E or with E only showed similar infectivity and titers in several cell lines and similar neutralizing sensitivity. These results showed that JEV-MuLV pseudotyped viruses did not require prM for production of infectious pseudotyped viruses.
Japanese encephalitis virus (JEV) is a serious mosquito-borne flavivirus that causes pandemic infectious disease of major public health importance in Asia. JEV is a member of the genusFlavivirus in the familyFlaviviridae, which includes yellow fever virus, Dengue virus, West Nile virus, and St. Louis encephalitis virus [1, 2].
The JEV single-stranded RNA genome (≈ 11 kb) encodes three structural proteins – capsid (C), premembrane (prM) or membrane (M), and envelope (E) protein – and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins [3–5].
The assembly of JEV in the endoplasmic reticulum is followed by modification of the two envelope proteins E and prM and virion export through the secretory pathway. PrM (≈ 26 kDa) is a precursor of the membrane-anchored and it cleaved a soluble Pr peptide and virion associated M protein (≈ 8 kDa) by trans-Golgi resident furin or related enzyme , resulting in two different forms of virion: the intracellular E- and prM-containing form, and the extracellular E- and M-containing form [3, 7].
The E protein plays a major role in virus assembly, adhesion, receptor binding and membrane fusion, hemagglutination inhibition (HI), and induction of neutralizing antibodies (Nabs) [8–10]. Therefore, the E protein is the principal target of neutralization by specific antibodies against JEV infection [4, 11]. E proteins of JEV expressed in different viral vector systems such as vaccinia virus, sindbis virus, and baculovirus have elicited high levels of neutralizing antibodies against JEV infection and have been tested as second generation JEV vaccines in mice [7, 12–14]. From these reports, it is unknown whether prM cleavage affects infectivity, E protein expression, or induction of neutralizing activity. A major function of prM was studied by blocking prM cleavage or by mutation of the conserved glycosylation motif of JEV prM . Even though the direct role of prM during the viral replication was not elucidated , it has a crucial function in multiple stages of JEV biology.
Pseudotyped viruses encoding prM/E or E of JEV NK and BJ strain were produced as previously described . Briefly, TELCeB6 cells, a MuLV packaging cell line , were transfected with pHCMV-E or pHCMV-prM/E by a calcium phosphate method. After overnight incubation, the culture medium was replaced, and the cells were incubated for two additional days. The supernatants containing pseudotyped viruses were harvested by low speed centrifugation (1,500 ×g, 5 min) to remove cell debris.
Infectivity and titer of JEV pseudotyped viruses in host cells
Infectious units/ml (IFU/ml)
JEV pseudotyed virus
The titers of pseudotyped viruses were comparable to the infectivity of JEV in each host cell line. JEV pseudotyped virus with E or prM/E (both NK and BJ strain) could efficiently infect several cell lines, with typical titers between 1.14 × 102 and 1.36 × 105 infectious units (IFU)/mL. From Table 1, viruses pseudotyped with prM/E exhibited infectivity similar to those pseudotyped with E only. This showed that the infectivity and titer of JEV pseudotyped virus is not affected by prM deletion.
Neutralizing sensitivity was tested with JEV-immunized immune sera, which was supplied by the Catholic University of Korea, to check the effect of prM on pseudotyped JEV antigens. As previously described , neutralization assays were carried out with Vero cells in triplicate. Approximately 100 IFU/mL of pseudotyped viruses were incubated with 10-fold diluted sera from mice immunized with JEV for 1 h at 37°C, and the mixture was subsequently added to Vero cells. After 2 days of incubation, virus infection was monitored by X-gal staining as described above. The neutralizing sensitivity was expressed as virus reduction by neutralizing antibodies.
In summary, we generated pseudotyped JEVs that express prM/E or E proteins from two JEV vaccine strains NK and BJ. All four JEV pseudotyped viruses efficiently infected several cell lines and were neutralized by sera from JEV-immunized mice. The main purpose of generating JEV pseudotyped virus was to devise a safe and rapid assay system to assess neutralizing antibodies by avoiding the use of infectious, replication-competent JEVs in a Biosafety Level 3 laboratory. The titer of the four JEV pseudotyped viruses was greater than 104 IFU/ml. This confirmed the possibility of mass-producing viruses to conduct neutralization assays with Vero and CRFK cells. Even though a crucial function of prM in assembly and maturation of flaviviruses has been reported, the two types of JEV-MuLV pseudotyped virus that respectively express the E or prM/E proteins were found to have no significant difference in the level of transcription and the extent of protein expression, infectivity, titer, and neutralization sensitivity.
This work was supported by a grant (20070501034009) from BioGreen 21 Program, Rural Development Administration and a Agricultural R&D Promotion Center, Ministry for Agriculture, Forestry and Fisheries, (608001-5), and a grant [K09040] from the Korea Institute of Oriental Medicine, Republic of Korea.
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