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. 2021 Sep 27;95(20):e0097321.
doi: 10.1128/JVI.00973-21. Epub 2021 Jul 28.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots

Mona Teppor  1 , Eva Žusinaite  1 , Liis Karo-Astover  1 , Ailar Omler  1 , Kai Rausalu  1 , Valeria Lulla  1 , Aleksei Lulla  1 , Andres Merits  1
Affiliations

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots

Mona Teppor et al. J Virol. .

Abstract

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.

Keywords: RNA replication; adaptive mutations; alphavirus; promoters; proteases; replicase.

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Figures

FIG 1
FIG 1
Swapping of fragments of SFV with homologous regions of CHIKV or SINV. (A) Schematic diagram of the SFV genome showing the domain organization of the nsP2-nsP3 region and the ns-regions of the chimeric genomes. NTD, N-terminal domain of nsP2; RecA, RecA-like domain; MTL, methyltransferase-like domain; AUD, alphavirus unique domain; HVD, hypervariable domain. The numbers indicate the amino acid residues flanking the swapped domains; sequences from CHIKV are shown in gray, and sequences from SINV are shown in black. The number of amino acid substitutions introduced by each of the swaps is shown, and the percent amino acid identity of the proteins encoded by the swapped regions is shown in parentheses. ICA titers are presented as PFU per 1 μg RNA and represent the averages of the data obtained in four independent experiments; <2 indicates values below the detection limit. Plaque sizes are indicated as follows: large, >3 mm; small, 1 to 2 mm; very small, <1 mm in diameter. P0 titers are presented as PFU per ml and represent the averages of the titers measured in three independent experiments. Please note that P0 stocks were harvested upon detection of CPE, i.e., at different times for different viruses. (B) Multistep growth curves of WT SFV, SFVCN, SFVCM, and SFVCA. BHK-21 cells were infected with P0 stocks of the indicated viruses at an MOI of 0.1. The culture medium was sampled immediately (0 h) and at 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h p.i., and the amount of infectious virus present was measured using a focus-forming assay. Each data point represents the mean ± standard deviation (SD) from three independent experiments. The dotted line indicates the limit of detection.
FIG 2
FIG 2
Adaptation of chimeric viruses results in nonsynonymous mutations in the ns-region. (A) Locations of nonsynonymous mutations found in adapted stocks of SFVCP2, SFVCP, SFVSN, and SFVSM. The numbers on the right sides of the drawings represent the number of virus isolates in which the indicated combinations of mutations were found. (B) The identified nonsynonymous mutations increase the infectivity of the swapped genomes. P0 stocks were harvested, and their ICA titers and P0 titers were determined and are shown in Fig. 1A. Titers of P1 stocks collected from infected C6/36 cells are presented as PFU per ml and represent the averages of the values obtained in three independent experiments.
FIG 3
FIG 3
In cellulo-evolved viruses with adaptive mutations display growth attenuation. (A and B) BHK-21 cells were infected at an MOI of 0.1 with WT SFV, with evolved stocks of (A) (SFVCP2, SFVSM) or (B) (SFVCP) or with P0 stocks of (A) (SFVCP2+2A796V, SFVSM+2V515E) or (B) (SFVCP+2V2F, SFVCP+2G460S, or SFVCP+4R611K). At 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h p.i., the growth medium was collected, and the virus in the medium was titrated using a focus-forming assay. Each data point represents the mean ± SD from three independent experiments. The dotted line indicates the limit of detection.
FIG 4
FIG 4
Swapping of the nsP2, nsP2 NTD, nsP2p, or nsP3 macrodomains often results in altered ns-polyprotein processing. In vitro translation was conducted using the TNT SP6 rabbit reticulocyte system, and plasmids containing icDNAs of the indicated viruses were used as templates. After 45 min, the reaction was stopped with cycloheximide, and samples were collected immediately (0′) or after incubation for an additional 30 min (30′). The produced ns-polyproteins and their processing products were separated using SDS-PAGE, and the densities of bands corresponding to nsP2 and P123 were quantified using ImageQuant software. (A) Translation of pSFVCP2 and its variants containing adaptive mutations. (B) Translation of other icDNAs harboring CHIKV-derived sequences. (C) Translation of icDNAs harboring SINV-derived sequences. The positions of P1234, P123, and P12/P23/P34 and those of these mature nsPs are shown. The ratio shows the amount of label that was incorporated into mature nsP2 relative to the amount that was incorporated into unprocessed P123; the nsP2/P123 ratio of WT SFV at 0′ was taken as 100. Each number represents the mean ± SD of the values obtained in three independent experiments. * indicates complete processing of P123 (corresponding density at background level). An additional band visible in samples of SFVCP2+4C483Y most likely represents C-terminally truncated P34. It was detected using only one batch of the TNT SP6 rabbit reticulocyte system and probably represents an artifact generated by premature termination of transcription in the region corresponding to the C483Y substitution.
FIG 5
FIG 5
Mutations in the P-side of the 1/2 site affect the rescue of SFVCP and SFVCP2 and the processing of the corresponding ns-polyproteins. (A) Sequences of the P6-P1′ regions of the 1/2 sites of CHIKV, SFV, SFVCP, and SFVCP2. Mutations introduced to obtain SFVCP+1G536V, SFVCP+1Y533D+1H534R, SFVCP2+1G536V, and SFVCP2+1Y533D+1H534R are shown in boldface and italics. (B) Localization of swapped regions and introduced mutations in the ns-regions of the chimeric genomes. ICA titers are shown as PFU per 1 μg RNA and represent the averages of the values obtained in three independent experiments; <2 indicates values below the detectable limit. Plaque sizes are indicated as follows: large, >3 mm; very small, <1 mm in diameter. P0 titers are shown as PFU per ml and represent the averages of the values obtained in three independent experiments. The data for WT SFV, SFVCP, and SFVCP2 are replotted from Fig. 1A. (C) Results of in vitro translation and processing. The assay was conducted as described in the legend to Fig. 4. Each number represents the mean ± SD from three independent experiments. * indicates complete processing of P123 (corresponding density at background level); the numbers in parentheses indicate a lack of increase in the amount of product with the same mobility as that of nsP2 during the chase.
FIG 6
FIG 6
Swaps and adaptive mutations introduced into the ns-region of SFV have different impacts on the activity of viral replicase. (A) Organization of the plasmids used for the trans-replication assay. CMV, immediate early promoter of human cytomegalovirus; LI, leader sequence of the herpes simplex virus thymidine kinase gene; SV40Ter, simian virus 40 late polyadenylation region. The position of the inactivating mutation in the nsP4 catalytic site of CMV-P1234GAA-SFV is indicated. HSPolI-FG-SFV contains the following: HSPolI, a truncated (−211 to −1) promoter for human RNA polymerase I; SFV 5′ UTR and partial 3′ UTR; N77, a region encoding the 77 N-terminal amino acid residues of nsP1; SG, the SFV SG promoter; RZ, an antisense-strand ribozyme of the hepatitis delta virus; and MmTer, a terminator for RNA polymerase I in mice. The drawings are not to scale. (B and C) U2OS cells in 12-well plates were cotransfected with 1 μg of HSPolI-FG-SFV and 1 μg of CMV-P1234-SFV or with a plasmid carrying the swaps and point mutations indicated on the horizontal axis. As a negative control, CMV-P1234GAA-SFV, which lacks polymerase activity, was used. The cells were incubated at 37°C and lysed at 16 h p.t. The activities of (B) Fluc, a proxy of replication, and (C) Gluc, a proxy of transcription, in the presence of the analyzed replicases were normalized to those of the P1234GAA controls. The means ± SD of the values obtained in four independent experiments are shown.
FIG 7
FIG 7
The presence of an adenine residue at position −1 of the SG promoter of SFV increases template transcription. (A) Organization of the plasmids used in the trans-replication assay. The substitution of the −1 guanine residue in the SG promoter with adenine, which yielded the HSPolI-FG-SFV-SG−1G/A template-expression plasmid, is shown in the drawing. The other designations are the same as in Fig. 6A. (B) U2OS cells in 12-well plates were cotransfected with 500 ng of HSPolI-FG-SFV or HSPolI-FG-SFV-SG−1G/A and with 500 ng CMV-P1234-SFV (WT) or with a plasmid carrying the swaps and point mutations indicated on the horizontal axis. As a negative control, CMV-P1234GAA-SFV, which lacks polymerase activity, was used. The cells were incubated at 37°C, and aliquots of the culture supernatants were collected at 16, 20, and 24 h p.t. The activities of Gluc produced in the presence of the analyzed replicases were normalized to those of the P1234GAA controls. The means ± SD of three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (Student’s unpaired t test).
FIG 8
FIG 8
Effect of swapping nsP2p and of adaptive mutations on NLuc expression and replication of SFV replicon vectors. (A) Schematic presentation of the SFV1-NLuc replicon, the chimeric SFV1CP-NLuc replicon, and SFV helper RNAs. Δ307-6400 indicates a deletion in the ns-region of helper RNAs. The adaptive mutations present in SFV1CP+2V2F-NLuc and SFV1CP+2G460S-NLuc replicons are shown. (B) BHK-21 cells were transfected with RNA transcripts corresponding to SP6-SFV1-NLuc (WT), SP6-SFV1CP-NLuc, SP6-SFV1CP+2V2F-NLuc, or SP6-SFV1CP+2G460S-NLuc replicons with or without transcripts corresponding to SFV helper RNAs. At 24 h p.t., the cells were harvested and lysed, and the NLuc activity present in an amount of lysate corresponding to 12,000 cells was measured; the value obtained for cells transfected with SP6-SFV1-Nluc (WT) transcript was taken as 100. The means ± SD of the values obtained in three independent experiments performed in triplicate are shown. ****, P < 0.0001; ns, not significant (Student’s unpaired t test). (C) Lysates of BHK-21 cells transfected with RNA transcripts corresponding to SP6-SFV1-NLuc (WT), SP6-SFV1CP-NLuc, SP6-SFV1CP+2V2F-NLuc, and SP6-SFV1CP+2G460S-NLuc replicons with or without transcripts corresponding to SFV helper RNAs and lysates from mock-transfected control cells were subjected to SDS-PAGE and immunoblot analysis using antibodies against SFV nsP1 (left panel) and SFV capsid protein (right panel). β-actin was used as a loading control. The detection of capsid protein expression was performed three times with similar results; data from one experiment are shown.
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