Differences in Processing Determinants of Nonstructural Polyprotein and in the Sequence of Nonstructural Protein 3 Affect Neurovirulence of Semliki Forest Virus

Abstract

The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV.

Importance: SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes were confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.

FIG 1

Recombinant constructs and viruses carrying two copies of nsP3-encoding sequences. (A and B) Schematic representations show the recombinant SFV constructs based on original rA774 and SFV4 sequences (A) and constructs based on corrected A774wt and SFV6 sequences containing codon-altered copy of nsP3 encoding region (B). (C and D) Results of ICA and titers of collected P₀ stocks. Multistep (C) and single-step (D) growth curves of recombinant viruses were determined. BHK-21 cells were infected at the indicated MOI, and samples were collected at the indicated time points. Data from one of two reproducible experiments are shown in each panel.

FIG 2

Localization of ns proteins in BHK-21 cells infected with SFV6 (A) and SFV6-6/6 (B). Cells were infected at an MOI of 10, fixed at 4 h p.i., permeabilized, and probed with antibodies for dsRNA (green) and nsP1, nsP2, nsP3, and nsP4 of SFV (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (for clarity, shown only on merge panels). Merged images of mock-infected cells (mock) stained using the same antibodies are shown for comparison.

FIG 3

Analysis of in vitro genetic stability, ns protein expression, and ns polyprotein processing of the viruses harboring two copies of nsP3. (A) Schematic representation of the assay and results of the RT-PCR analysis of the region containing duplicated nsP3-encoding sequences. RNA obtained from P₀ (left panel), P₃ (middle panel), and P₅ (right panel) stocks was reverse transcribed, cDNA was PCR amplified using primers corresponding to the 3′ region of the nsP2-encoding sequence and complementary to the 5′ region of the native nsP3-encoding sequence. PCR products were analyzed on a 1% agarose gel. (B) BHK-21 cells infected at an MOI of 1 were collected 8 h p.i. and lysed, and SFV ns proteins were analyzed using SDS-PAGE. nsPs were detected using corresponding polyclonal antisera; their positions and that of P34 are shown on the right side of the panel. β-Actin was used as a loading control. (C) Processing of ns polyproteins in BHK-21 cells. Cells were infected with SFV6, SFV6-6/74, or SFV6-6/6 at an MOI of 10, starved for 30 min at 3 h p.i. in methionine-cysteine-free medium, and then pulsed with 50 μCi of [³⁵S]methionine-cysteine mixture for 15 min. Cells were collected immediately after pulse or after a 45-min chase. Synthesized ns polyproteins, their processing intermediates, and mature nsPs were immunoprecipitated using the indicated combinations of antibodies. Positions of nsPs and ns polyproteins are shown at both sides of the panel. For all panels, data from one of three reproducible experiments are shown.

FIG 4

Neurovirulence of SFV6, A774wt, and viruses harboring two copies of nsP3. (A) Groups (n = 37) of 4- to 5-week-old mice were inoculated i.p. with the indicated virus (5,000 PFU/mouse) and monitored for survival for 10 days. (B) Groups (n = 7) of 4- to 5-week-old mice were inoculated i.p. with the indicated virus (5,000 PFU/mouse) and monitored for survival for 10 days. (C and D) Virus titers in the blood of infected animals on PID 1 (C) and on PID 3 (D). (E) Virus titers in brain tissue on PID 3. Each symbol represents an individual mouse; the line depicts the mean titer of each group. The dotted line depicts the limit of detection. Statistical significance was determined using the Mann-Whitney test. Only statistically significant differences are indicated: **, P < 0.01; ****, P < 0.0001. (F) Detection of viruses with two copies of nsP3 in brains of mice at the time of the clinical endpoint of the experiment. Total RNA was isolated from brain homogenates, reverse transcribed, and analyzed using PCR. The left panels show schematic representations of PCR products. Obtained PCR products were analyzed on a 1% agarose gel (right panels).

FIG 5

Swapping of nsP3 regions between SFV6 and A774wt results in virulent chimeras. (A) Schematic representations of the SFV constructs, the results of ICA, and titers of P₀ virus stocks. (B) Groups (n = 7) of 4- to 5-week-old mice were inoculated i.p. with 5,000 PFU of the indicated virus (mice infected with SFV6 are the same as in Fig. 4B) and monitored for survival for 10 days. (C and D) Virus titers in the blood on PID 1 (C) and on PID 3 (D). (E) Virus titers in brain tissue on PID 3. Each symbol represents the result for an individual mouse. The line depicts the mean titer of each group. The dotted line depicts the limit of detection. Significance was determined using the Mann-Whitney test. Only statistically significant differences are indicated: *, P < 0.05; **, P < 0.01.

FIG 6

Mutations at positions associated with ns polyprotein processing affect SFV neurovirulence. (A) Schematic representations of the recombinant SFV constructs and the results of ICA and titers of P₀ virus stocks. (B) Groups (n = 7) of 4- to 5-week-old mice were inoculated i.p. with 5,000 PFU of the indicted virus (note that the mice infected with SFV6 are different from those shown in Fig. 4 and 5) and monitored for survival for 10 days. (C and D) Virus titers in the blood on PID 1 (C) and PID 3 (D). (E and F) Virus titers in brain tissue on PID 1 (E) and PID 3 (F). Each symbol represents an individual mouse. The line depicts the mean titer of each group. The dotted line depicts the limit of detection. Differences between brain titers of A774wt-HV at PID 1 (E) and at PID 3 (F) as well as differences between brain titers of SFV6-74-RE at PID 1 (E) and PID 3 (F) are statistically significant (P = 0.008 and P = 0.02, respectively).

FIG 7

Changes at the P4 position of the 1/2 site and at the S4 subsite residue of nsP2 alter the speed of ns polyprotein processing in infected cells. (A) BHK-21 cells were infected at an MOI of 10 with A774wt, A774wt-HV, SFV6-74, or SFV6-74-RE. At 3 h p.i., cells were starved in methionine-cysteine-free medium and then pulsed with 50 μCi of [³⁵S]methionine-cysteine mixture for 15 min. Cells were collected immediately after pulse or after 45-min chase. Synthesized ns polyproteins, their processing intermediates, and mature nsPs were immunoprecipitated using antibodies against the indicated proteins. The positions of P123, P34, and nsP2 are indicated with arrows at the left side of the panel. Data from one of three reproducible experiments are shown. (B) Quantification of data obtained in three independent pulse-chase experiments. The ratio of P123 to nsP2 was determined as the ratio of label present in the corresponding products. Columns represent the averages of three experiments; error bars represent standard errors of the means.