Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Abstract

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

FIG. 1.

Schematic representation of replicons and their ability to persistently replicate in BHK-21 cells. A detailed description of the replicon constructs is presented in Materials and Methods. Arrows indicate the positions of the subgenomic promoters. The positions of the mutations in the VEE 5′ UTR and SIN nsP2 coding gene are indicated. BHK-21 cells were transfected by 4 μg of each in vitro-synthesized replicon RNA using electroporation, and different dilutions of the cells were seeded into 100-mm dishes. Puromycin selection (10 μg/ml) was applied 24 h posttransfection. After 5 days of incubation under puromycin, dishes containing 1% of electroporated cells were stained with crystal violet. The efficiency of focus formation was calculated based on the number of foci in the dishes containing fewer electroporated cells.

FIG. 2.

Analysis of RNA replication and transcription of the subgenomic RNA in the cells transfected with different replicons. (A) BHK-21 cells were transfected with 4 μg of the in vitro-synthesized replicons' RNA, and equal numbers of electroporated cells (∼10⁶ cells) in six-well Costar plates were labeled with [³H]uridine (20 μCi/ml) in the presence of dactinomycin (1 μg/ml) between 4 and 8 h posttransfection. RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes contain RNAs from 3 × 10⁵ cells. The gel was fluorographed and exposed for 60 h. Levels of replicon genome RNA accumulation relative to that of the SINrep/Pac replicon and molar ratio of the subgenome-to-genome RNA synthesis were determined by excision of radiolabeled bands and liquid scintillation counting. N.D indicates that RNA replication was below the detection limit of the procedure applied. (B) RNA replication in the cells containing persisting replicons. After 6 days of selection with puromycin, 10⁶ Pur^r BHK-21 cells containing different replicons were labeled with [³H]uridine (20 μCi/ml) in the presence of dactinomycin (1 μg/ml) for 8 h. RNAs were isolated and analyzed by agarose gel electrophoresis. Lanes contain RNAs from 3 × 10⁵ cells. The gel was fluorographed and exposed for 6 days. G and SG indicate positions of replicons' genomic and subgenomic RNAs, respectively.

FIG. 3.

Schematic representation of viral genomes and analysis of virus growth. (A) Both genomes encoded the same nonstructural proteins derived from VEE TC-83. 5′VEE/SIN had a 5′ UTR derived from the VEE TRD strain. A detailed description of the chimeric viral genome is presented in Materials and Methods. Solid boxes and open boxes indicate VEE genome- and SIN genome-derived sequences, respectively. (B) BHK-21 cells in six-well Costar plates (5 × 10⁵ cells/well) were infected with the indicated viruses at an MOI of 1 PFU/cell. At the indicated times, the media were replaced, and virus titers were determined as described in Materials and Methods.

FIG. 4.

Schematic representation of the double subgenomic replicons expressing Pac and GFP or SEAP and analysis of protein expression. (A) The designed replicons encoded the nonstructural proteins and homologous cis-acting elements (5′ UTR, 3′ UTR, subgenomic promoters, and 5′ UTRs in the subgenomic RNAs) derived from VEE, EEE, and SIN viruses (see Materials and Methods for details). All of the constructs had one subgenomic promoter driving the expression of the Pac gene and a second promoter driving the expression of either GFP or SEAP. Mutations in the 5′ UTR of the VEE TC-83-based replicons and in the nsP2 gene of the SIN-based replicon are indicated. (B) Analysis of alkaline phosphatase expression in the cells carrying persistently replicating VEE and EEE replicons. We seeded 2 × 10⁵ replicon-containing cells into six-well Costar plates. After 4 h of incubation at 37°C, cells were washed with phosphate-buffered saline and further incubated in 2 ml of complete growth medium. At the indicated time points, 200-μl aliquots of medium were taken, and the same volume was added to the wells. Alkaline phosphatase activity was analyzed as described in Materials and Methods. (C) Analysis of GFP expression in the cells carrying persistently replicating VEE, EEE, and SIN replicons with adaptive mutation in nsP2. Cells were analyzed without fixation by flow cytometry on a FACS Vantage (Becton Dickinson).

FIG. 5.

Stability of GFP expression by the persistently replicating EEErep/GFP/Pac replicon. Previously selected Pur^r cells were passaged (1:10, approximately every 48 h) in the absence of puromycin in the medium. At the indicated times, the percentage of GFP-negative cells was calculated by examination of several random fields on the inverted fluorescent microscope. Puromycin was added back to the medium after 18 days of passaging in drug-free medium.

FIG. 6.

Analysis of replicons' interference with different viral infections. Cells carrying persistently replicating EEErep/Pac or 5′VEErep/Pac replicons were infected with RVFV MP12 (upper left panel), WNV (upper right panel), VEE TC-83 and SIN Toto1101 (lower left panel), and EEE NA Florida 91 and SIN Toto1101 (lower right panel). BHK-21 or Pur^r cells (5 × 10⁵) carrying the indicated replicons in six-well Costar plates were infected with viruses at the MOIs indicated in the figure. At the indicated times, media were replaced and virus titers were determined as described in Materials and Methods.

FIG. 7.

Sequence alignment and adaptive mutations detected in the nsP2 and nsP3 coding sequence of VEE replicons (panels A and B, respectively) and the mutations found in other alphaviruses and replicons that affect their ability to cause CPE. VEE, Venezuelan equine encephalitis virus (21); EEE, eastern equine encephalitis virus (41); SIN, Sindbis virus; SFV, Semliki Forest virus (40). Residues identical to those in the VEE sequence are indicated by dashes. All of the mutations are highlighted. The mutated amino acids in viral proteins are indicated by black boxes. Corresponding amino acids in the proteins of other alphaviruses are indicated by shaded boxes.

FIG. 8.

Analysis of replicons' efficiency to persistently replicate in different cell lines. (A) Huh-7 cells were transfected with 12 μg of 5′VEErep/Pac RNA, followed by puromycin selection. Colonies of Pur^r cells were stained after 20 days of drug treatment. (B) Huh-7 cells were transfected with 12 μg of 5′VEErep/S/Pac RNA (containing the P₇₇₃→S mutation in nsP2) and treated with puromycin for 10 days. Wedetected no cell death due to CPE caused by replication of virus-specific RNAs or drug treatment. (C) Different Pur^r, GFP-expressing cell lines were generated by transfection of 4 μg of 5′VEErep/S/GFP/Pac RNA followed by puromycin selection. (D) BHK-21 cells were transfected with 4 μg of in vitro-synthesized replicons' RNA, and equal numbers of electroporated cells (∼10⁶ cells) in six-well Costar plates were labeled with [³H]uridine (30 μCi/ml) in the presence of dactinomycin (1 μg/ml) at 3 to 7 h posttransfection. RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes contain RNAs from 2.5 × 10⁵ cells. G and SG indicate positions of replicons' genomic and subgenomic RNAs, respectively.