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. 2023 May 31;97(5):e0058523.
doi: 10.1128/jvi.00585-23. Epub 2023 May 10.

The Reovirus σ1 Attachment Protein Influences the Stability of Its Entry Intermediate

Maximiliano L Garcia  1 , Pranav Danthi  1
Affiliations

The Reovirus σ1 Attachment Protein Influences the Stability of Its Entry Intermediate

Maximiliano L Garcia et al. J Virol. .

Abstract

Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.

Keywords: assembly; capsid; reovirus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
T3DF/T3DCS1, T3DF/S1-22C, and T3DF/S1-408C ISVPs are less stable than T3DF. (A) ISVPs (2 × 1012 particles/mL) of the indicated virus strains were incubated at the indicated temperatures for 5 min. After heat treatment, the aliquots were incubated with 0.1 mg/mL trypsin for 30 min on ice. Loading dye was added and samples were heated at 95°C for 5 min. Samples were resolved on 10% SDS-PAGE gels. These gels represent at least three independent experiments. (B) ISVPs of the indicated virus strains were heated at a range of temperatures. After heating, the samples were subjected to plaque assay. Change in titer (represented as log10 PFU/mL) at each temperature relative to 4°C was measured. Data are representative of three independent experiments. Error bars represent SD of three replicates. Two-way ANOVA with Dunnett’s multiple comparisons was performed. **, P < 0.001; ***, P < 0.0005; ****, P < 0.0001.
FIG 2
FIG 2
Residues in the head and tail domains of T3D σ1 impact stability and encapsidation. (A) Purified virions of the indicated virus strains were diluted in 0.1M carbonate-bicarbonate buffer and coated overnight on to high-affinity-binding polystyrene plates at 4°C. After blocking, the virus was stained with reovirus specific rabbit polyclonal antiserum or σ1 specific 9BG5 MAb. After labeling with appropriate secondary antibodies the plate was imaged on LI-COR Odyssey scanner. The results are plotted as the ratio of 9BG5 intensity compared to reovirus polyclonal intensity in the same well. 3 wells were counted and error bars represent SD. *, P < 0.05 as determined by one-way ANOVA with Dunnett’s multiple-comparison test. Samples were compared to T3DF (B) 1 × 1011 virions were resolved on an ultra-pure agarose gel based on their σ1 trimer content. After colloidal blue staining, the gel was imaged on a LI-COR Odyssey scanner. (C) The percentage of particles present in each species was quantified relative to the total intensity of one lane. (D) L929 cells were infected with the indicated virus strain at an MOI of 5 PFU/cell. An in-cell western was completed using MAb 9BG5 (for σ1) or anti-reovirus antisera. After labeling with appropriate secondary antibodies the plate was imaged on LI-COR Odyssey scanner. The results are plotted as the ratio of 9BG5 intensity compared to reovirus polyclonal intensity. Mean ratios of three different infections and SD are plotted. (E) Differences are not statistically significant.
FIG 3
FIG 3
Tail and body domains of σ1 influence ISVP stability. (A) Schematic representation of type 1 virus (T1), type 3 virus (T3), and chimeric σ1 proteins. (B) T1L ISVPs (2 × 1012 particles/mL) expressing various σ1 proteins were incubated at the indicated temperatures for 5 min. After heat treatment, the aliquots were incubated with 0.1 mg/mL trypsin for 30 min on ice. Loading dye was added and samples were heated at 95°C for 5 min. Samples were resolved on 10% SDS-PAGE gels. These gels represent at least three independent experiments.
FIG 4
FIG 4
Alterations in σ1 tail and body domains affect its encapsidation on particles. (A) Purified virions expressing the indicated σ1 were diluted in 0.1M carbonate-bicarbonate buffer and coated overnight on to high-affinity-binding polystyrene plates at 4°C. After blocking, the virus was stained with reovirus specific rabbit polyclonal antiserum or σ1 specific 5C6 MAb. After labeling with appropriate secondary antibodies the plate was imaged on LI-COR Odyssey scanner. The results are plotted as the ratio of 5C6 intensity compared to reovirus polyclonal intensity in the same well. Three wells were counted and error bars represent SD. *, P < 0.05 as determined by one-way ANOVA Dunnett’s with multiple comparisons. Samples were compared to T1L (B) 1 × 1011 virions were resolved on an ultra-pure agarose gel based on their σ1 trimer content. After colloidal blue staining, the gel was imaged on LI-COR Odyssey scanner. (C) The percentage of particles in each trimer species was quantified relative to the total. (D) There are no replicates so we cannot determine significance values.
FIG 5
FIG 5
σ1 encapsidation level of wild-type virus affects ISVP stability. (A) Diagram of T1L separated based on encapsidation status. (B) 1 × 1011 virions were resolved on an ultra-pure agarose gel based on their σ1 trimer content. The same sample was run on multiple lanes. One lane was cut and stained for 3 h with ethidium bromide. Using the stained lane as guide, the other lanes were cut into groups based on the amount of encapsidated σ1. Samples were minced, sonicated, and nutated. The released virus was converted to ISVPs and heated at a range of temperatures. After heating, the samples were subjected to plaque assay. The titer (represented as log10 PFU/mL) at each temperature relative to 4°C was measured. Data are representative of three independent experiments.
FIG 6
FIG 6
Selection of thermostable revertant, T3DF/T3DCS1-T132A. (A) Candidate thermostable revertants (heat resistant [HR] were converted to ISVPs and incubated at 4°C or 44°C). After incubation the samples were subjected to plaque assay to determine the titer. Change in titer (represented as log10 PFU/mL) at 44°C relative to 4°C was measured. (B and C) UCSF Chimera rendered image of a side view (B) or top view (C) of the σ1 trimer. PDB accession number (6GAP) (2OJ5). The position of the mutated threonine at residue 132 is highlighted by green spheres.
FIG 7
FIG 7
σ1 of T132A restores T3DF/T3DCS1 ISVP stability without enhancing encapsidation of σ1. (A) ISVPs (2 × 1012 particles/mL) of the indicated virus strains were incubated at the indicated temperatures for 5 min. After heat treatment, the aliquots were incubated with 0.1 mg/mL trypsin for 30 min on ice. Loading dye was added and samples were heated at 95°C for 5 min. Samples were run on SDS-PAGE gels. These gels represent at least three independent experiments. (B) Purified virions of the indicated virus strains were diluted in 0.1M carbonate-bicarbonate buffer and coated overnight on to high-affinity-binding polystyrene plates at 4C. After blocking, the virus was stained with reovirus specific rabbit polyclonal antiserum or s1 specific 9BG5 MAb. After labeling with appropriate secondary antibodies the plate was imaged on LI-COR Odyssey scanner. The results are plotted as the ratio of 9BG5 intensity compared to reovirus polyclonal intensity in the same well. 3 wells were counted and error bars represent SD. (C) 1 × 1011 virions were resolved on an ultra-pure agarose gel based on their σ1 trimer content. After colloidal blue staining, the gel was imaged on LI-COR Odyssey scanner.
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References

    1. Wodrich H, Guan T, Cingolani G, Von Seggern D, Nemerow G, Gerace L. 2003. Switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals. EMBO J 22:6245–6255. doi:10.1093/emboj/cdg614. - DOI - PMC - PubMed
    1. Wiethoff CM, Wodrich H, Gerace L, Nemerow GR. 2005. Adenovirus protein VI mediates membrane disruption following capsid disassembly. J Virol 79:1992–2000. doi:10.1128/JVI.79.4.1992-2000.2005. - DOI - PMC - PubMed
    1. Wodrich H, Henaff D, Jammart B, Segura-Morales C, Seelmeir S, Coux O, Ruzsics Z, Wiethoff CM, Kremer EJ. 2010. A capsid-encoded PPxY-motif facilitates adenovirus entry. PLoS Pathog 6:e1000808. doi:10.1371/journal.ppat.1000808. - DOI - PMC - PubMed
    1. Schreiner S, Martinez R, Groitl P, Rayne F, Vaillant R, Wimmer P, Bossis G, Sternsdorf T, Marcinowski L, Ruzsics Z, Dobner T, Wodrich H. 2012. Transcriptional activation of the adenoviral genome is mediated by capsid protein VI. PLoS Pathog 8:e1002549. doi:10.1371/journal.ppat.1002549. - DOI - PMC - PubMed
    1. Moyer CL, Besser ES, Nemerow GR. 2016. A single maturation cleavage site in adenovirus impacts cell entry and capsid assembly. J Virol 90:521–532. doi:10.1128/JVI.02014-15. - DOI - PMC - PubMed

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