Reovirus Core Proteins λ1 and σ2 Promote Stability of Disassembly Intermediates and Influence Early Replication Events

Abstract

The capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is composed of μ1-σ3 heterohexamers which surround the core. The core is composed of λ1 decamers held in place by σ2. After entry into the endosome, σ3 is proteolytically degraded and μ1 is cleaved and exposed to form infectious subvirion particles (ISVPs). ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of μ1 peptides, which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work identified regions or specific residues within reovirus outer capsid proteins that impact the efficiency of cell entry. We examined the functions of the core proteins λ1 and σ2. We generated a reovirus T3D reassortant that carries strain T1L-derived σ2 and λ1 proteins (T3D/T1L L3S2). This virus displays lower ISVP stability and therefore converts to ISVP*s more readily. To identify the molecular basis for lability of T3D/T1L L3S2, we screened for hyperstable mutants of T3D/T1L L3S2 and identified three point mutations in μ1 that stabilize ISVPs. Two of these mutations are located in the C-terminal ϕ region of μ1, which has not previously been implicated in controlling ISVP stability. Independent of compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells than T3D. In addition to identifying a new role for the core proteins in disassembly events, these data highlight the possibility that core proteins may influence multiple stages of infection.IMPORTANCE Protein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. In comparison to the parental T3D strain, we found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of the virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

Keywords: capsid; double-stranded RNA virus; reovirus.

FIG 1

Schematic representation of reovirus capsid proteins.

FIG 2

T3D/T1L L3S2 exhibits increased efficiency of ISVP-to-ISVP* conversion in vitro. (A) T3D and T3D/T1L L3S2 virions (2 × 10¹² particles/ml) were divided into aliquots of equal volume and incubated either at 4°C or over a range of temperatures (65 to 85°C) for 5 min. The reaction mixtures were chilled on ice and digested with 0.10 mg/ml trypsin for 30 min. Following addition of loading dye, the samples were subjected to SDS-PAGE analysis. The positions of major capsid proteins are shown. μ1 runs as μ1C (15). (B) ISVPs (2 × 10¹¹ particles/ml) of T3D or T3D/T1L L3S2 were divided into aliquots of equivalent volume and incubated either at 4°C or over a range of temperatures (22 to 42°C) for 20 min. The reactions were chilled on ice and digested with 0.10 mg/ml trypsin for 30 min. Following addition of loading dye, the samples were subjected to SDS-PAGE analysis. The gels shown are representative of at least 3 independent experiments. The positions of major capsid proteins are shown. μ1 runs as μ1C. (C) ISVPs generated from P2 stocks of the indicated virus strain were divided into aliquots of equivalent volume and incubated at either 4°C or 40°C for 20 min. Reactions were then diluted in PBS and subjected to plaque assay. The data are plotted as mean loss of infectivity for three independent samples in comparison to samples incubated at 4°C. Error bars indicate SD. ***, P < 0.001 in comparison to T3D, as determined by Student's t test.

FIG 3

Increased ISVP-to-ISVP* conversion efficiency in T3D/T1L L3S2 is not due to altered interactions with viral RNA. ISVPs (2 × 10¹¹ particles/ml) derived from genome-containing or genome-deficient particles of strain T3D were divided into aliquots of equivalent volume and incubated either at 4°C or over a range of temperatures (22 to 40°C) for 20 min. The reaction mixtures were chilled on ice and digested with 0.10 mg/ml trypsin for 30 min. Following addition of loading dye, the samples were subjected to SDS-PAGE analysis. The positions of major capsid proteins are shown. μ1 runs as μ1C.

FIG 4

Selection of viruses with mutations that confer stability to T3D/T1L L3S2 ISVPs. (A) Diagram depicting the process for selecting for mutants with reduced ISVP-ISVP* conversion efficiency of T3D/T1L L3S2. ISVPs of T3D/T1L L3S2 were incubated at 40°C for 20 min. Reaction mixtures were then diluted in PBS and subjected to plaque assay. Viruses from resulting plaques were isolated and propagated to generate P0 stocks. Heat resistance of these putative heat-resistant (HR) plaque isolates was confirmed by measuring the thermal stability of ISVPs incubated at 4°C or 40°C using a plaque assay. Mutants that were confirmed as heat resistant were sequenced. (B) ISVPs generated from P0 stocks were incubated at either 4°C or 40°C for 20 min. Reaction mixtures were then diluted in PBS and subjected to plaque assay. Note that HR1 to -10 and HR11 to -20 are from two separate isolation experiments, collected and tested at different times. ND, not detectable. (C and D) Top (left) and side (right) views of the μ1 trimer (C) and monomer (D). Positions of mutations identified in HR viruses are shown in green. μ1 cleavage fragments are colored as in panel E, with one μ1 monomer shown with darker colors.

FIG 5

Mutations in μ1 restore stability. (A) ISVPs (2 × 10¹¹ particles/ml) of T3D/T1L L3S2 with the indicated M2 mutations were divided into aliquots of equivalent volume and incubated at either 4°C or over a range of temperatures (22 to 42°C) for 20 min. The reaction mixtures were chilled on ice and digested with 0.10 mg/ml trypsin for 30 min. Following addition of loading dye, the samples were subjected to SDS-PAGE analysis. The gels shown are representative of at least 3 independent experiments. The positions of major capsid proteins are shown. μ1 runs as μ1C. (B) ISVPs generated from P2 stocks of the indicated virus strain were divided into aliquots of equivalent volume and incubated at either 4°C or 40°C for 20 min. Reaction mixtures were then diluted in PBS and subjected to plaque assay. The data are plotted as mean loss of infectivity for three independent samples in comparison to samples incubated at 4°C. Error bars indicate SD. **, P < 0.01, and ***, P < 0.001, in comparison to T3D/T1L L3S2, as determined by Student's t test.

FIG 6

Mutations in μ1 hyperstabilize T3D. (A) ISVPs (2 × 10¹¹ particles/ml) of T3D and T3D with the indicated M2 mutations were divided into aliquots of equivalent volume and incubated at either 4°C or over a range of temperatures (32 to 46°C) for 20 min. The reaction mixtures were chilled on ice and digested with 0.10 mg/ml trypsin for 30 min. Following addition of loading dye, the samples were subjected to SDS-PAGE analysis. The gels shown are representative of at least 3 independent experiments. The positions of major capsid proteins are shown. μ1 runs as μ1C. (B) ISVPs generated from purified virions were divided into aliquots of equivalent volume and incubated at either 4°C or 49°C for 20 min. Reaction mixtures were then diluted in PBS and subjected to plaque assay. The data are plotted as mean loss of infectivity for three independent samples in comparison to samples incubated at 4°C. Error bars indicate SD. ***, P < 0.001, in comparison to T3D, as determined by Student’s t test.

FIG 7

T3D/T1L L3S2 affects viral replication. (A) L cell monolayers were infected with T3D or T3D/T1L L3S2 or with the indicated mutant viruses at an MOI of 0.1 PFU/cell. At 0 and 24 h postinfection, the infected cells were lysed and the viral yield was quantified by plaque assay. Error bars indicate SD. *, P < 0.05, and ***, P < 0.001, in comparison to T3D, as determined by Student's t test. Data are representative of at least 3 different experiments. (B) L cell monolayers were infected with the indicated viruses at an MOI of 10 PFU/cell. At 10 h postinfection, the cells were lysed and protein production was determined by immunoblotting. Protein quantification of 3 replicates normalized to PSTAIR is shown. Error bars indicate SD. ***, P < 0.001, in comparison to T3D, as determined by Student's t test. (C) L cell monolayers were infected with the indicated viruses at an MOI of 10 PFU/cell. The cells were lysed at 6 h postinfection, and total RNA was isolated. cDNA was generated using primers for T3D S1 and GAPDH. mRNA production was measured by RT-qPCR. Data are fold change compared to mock-infected samples and normalized to GAPDH. Error bars indicate SD. ***, P < 0.001, in comparison to T3D, as determined by Student's t test.