Venezuelan equine encephalitis virus nsP2 protein regulates packaging of the viral genome into infectious virions

Abstract

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein. The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

Fig 1

Mutations in CP-specific helix I have a positive impact on infectious titers of VEEV encoding CP with mutated RNA-binding domain. (A) Schematic representation of the VEEV genome encoding wt and mutated CP. The amino acid sequences of helix I and putative helix 2 are presented. The residual positively charged amino acids, which were not mutated in VEEV/Cm/GFP are indicated with blue letters. The adaptive N47Y mutation, identified in helix I of the variant capable of developing spreading infection, is indicated. (B) BHK-21 cells were electroporated with 4 μg of in vitro-synthesized RNA of VEEV/GFP and VEEV/Cm/GFP variants. Media were replaced at the indicated time points, and titers of infectious viral particles were determined as described in Materials and Methods. (C) BHK-21 cells were electroporated with 4 μg of in vitro-synthesized RNA of VEEV/Cm/GFP and VEEV/Cm1/GFP variants. Media were replaced at the indicated time points, and titers of infectious viral particles were determined as described in Materials and Methods. The dashed line indicates the limit of detection.

Fig 2

Adaptive mutations in VEEV nsP2 protein increase the release of infectious virions from cells containing replicating viral genomes encoding mutated CP. (A) Mutations identified in cytopathic, plaque-purified variants of passaged VEEV/Cm1/GFP. (B) Schematic representation of the VEEV nsP2 domain structure and positions of the identified mutations. (C and D) The schematic representation of recombinant VEEV/Cm1/GFP variant genomes with the indicated adaptive mutations in nsP2 and CP and their replication rates. BHK-21 cells were electroporated with 4 μg of in vitro-synthesized RNAs of the indicated variants. Media were replaced at the indicated time points, and titers of infectious viral particles were determined as described in Materials and Methods.

Fig 3

Adaptive mutations in nsP2 make VEEV variants encoding mutated CP more cytopathic. The schematic representation of VEEV genomes encoding wt and mutated nsP2 and CP. Mutant genomes were packaged into infectious virions as described in Materials and Methods, and these stocks were titrated on Vero cells. Plaques were stained with crystal violet after 3 days of incubation at 37°C.

Fig 4

Adaptive mutations in nsP2 can increase infectious titers of VEEV CP mutant independently of the CP-specific adaptive mutations. (A) Schematic representation of the genomes of VEEV CP mutants used in these experiments. The introduced adaptive mutations are indicated. (B) Viral genomes containing indicated mutations were packaged to high titers into infectious viral particles, and 5 × 10⁵ Vero cells in six-well Costar plates were infected at an MOI of 20 inf.u./cell as described in Materials and Methods. At the indicated times, media were replaced, and infectious titers were determined by plaque assay on Vero cells.

Fig 5

The nsP2-specific adaptive mutations identified make VEEV TC-83-based replicons more cytopathic. (A) Schematic representation of VEEV replicons encoding either wt or mutated nsP2 and containing GFP and Pac genes under the control of the subgenomic promoters. Equal amounts of the in vitro-synthesized RNAs were electroporated into BHK-21 cells, and the indicated colony-forming efficiencies were measured as described in Materials and Methods. (B) Representative dishes, in which equal numbers of electroporated cells were seeded. Colonies of replicon-containing cells were stained with crystal violet after 6 days of incubation at 37°C in the presence of puromycin at a concentration of 5 μg/ml.

Fig 6

Adaptive mutations in nsP2 increase synthesis of virus-specific RNAs and structural proteins. (A) The schematic representation of VEEV genomes containing adaptive mutations in CP and nsP2. (B) The indicated genomes were packaged to high titers into infectious viral particles as described in Materials and Methods, and Vero cells were infected at an MOI of 20 inf.u/cell. Viral RNAs were metabolically labeled with [³H]uridine between 3 and 7 h postinfection and analyzed as described in Materials and Methods. (C) Vero cells were infected as described above, and proteins were metabolically labeled with [³⁵S]methionine at 6 h postinfection and analyzed as described in Materials and Methods. This experiment was repeated twice with very similar results.

Fig 7

Adaptive mutations in nsP2, but not in CP, have negative effects on IFN-β induction. NIH 3T3 cells were infected with the indicated viruses as described in the Fig. 6 legend (see also in Fig. 6A the schematic representation of their genomes). Media were harvested at 24 h postinfection, and concentrations of IFN-β were measured as described in Materials and Methods. The experiment was repeated twice with very similar results.

Fig 8

Further virus evolution leads to a decrease in SVP production. (A) Schematic representation of VEEV genomes containing adaptive mutations in CP and nsP2. (B) Mutant genomes were packaged into infectious virions as described in Materials and Methods, and these stocks were titrated on Vero cells. Plaques were stained with crystal violet after 3 days of incubation at 37°C. (C) Vero cells in six-well Costar plates were infected with the indicated viruses at an MOI of 20 inf.u/ml. At the indicated times, media were replaced, and infectious titers were determined by plaque assay on Vero cells. Titers of VEEV/Cm were determined by immunostaining, using mouse antibodies, specific to VEEV structural proteins. (D) Vero cells were infected with the indicated mutants at an MOI of 20 inf.u/cell. Viral RNAs were metabolically labeled with [³H]uridine between 3 and 7 h postinfection and analyzed as described in Materials and Methods. (E) Vero cells were infected as described above, and proteins were metabolically labeled with [³⁵S]methionine at 6 h postinfection and analyzed as described in Materials and Methods. (F) Vero cells were infected with the indicated viruses as described above. At 6 h postinfection, media were replaced by serum-free VP-SFM media, and the released particles were harvested at 20 h postinfection. They were pelleted by ultracentrifugation and samples corresponding to 1 ml of media were analyzed by SDS–10%PAGE, followed by Western blotting with VEEV-specific antibodies. Quantitative analysis was performed on a LI-COR imager. The experiments were repeated three times with reproducible results. This figure represents one of the experiments.