scholarly journals Calicivirus VP2 forms a portal to mediate endosome escape

2018 ◽  
Author(s):  
Michaela Conley ◽  
Marion McElwee ◽  
Liyana Azmi ◽  
Mads Gabrielsen ◽  
Olwyn Byron ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Host Cells ◽  
Major Step ◽  
Endosomal Membrane ◽  
Endosome Escape ◽  
Animal Pathogens ◽  
Capsid Shell ◽  
Capsid Protein Vp1

AbstractTo initiate the infectious process, many viruses enter their host cells by triggering endocytosis following receptor engagement. The mechanism by which non-enveloped viruses, such as the caliciviruses, escape the endosome is however poorly understood. TheCaliciviridaeinclude many important human and animal pathogens, most notably norovirus, the cause of winter vomiting disease. Here we show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal assembly at a unique three-fold symmetry axis following receptor engagement. This feature surrounds an open pore in the capsid shell. We hypothesise that the VP2 portal complex is the means by which the virus escapes the endosome, pene-trating the endosomal membrane to release the viral genome into the cytoplasm. Cryogenic electron microscopy (cryoEM) and asymmetric reconstruction were used to investigate structural changes in the capsid of feline calicivirus (FCV) that occur when the virus binds to its cellular receptor junctional adhesion molecule-A (fJAM-A). Near atomic-resolution structures were calculated for the native virion alone and decorated with soluble receptor fragments. We present atomic models of the major capsid protein VP1 in the presence and absence of fJAM-A, revealing the contact interface and conformational changes brought about by the interaction. Furthermore, we have calculated an atomic model of the portal protein VP2 and revealed the structural changes in VP1 that lead to pore formation. While VP2 was known to be critical for the production of infectious virus, its function has been hitherto undetermined. Our finding that VP2 assembles a portal that is likely responsible for endosome escape represents a major step forward in our understanding of both theCaliciviridaeand icosahedral RNA containing viruses in general.

scholarly journals Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

2018 ◽  
Vol 92 (11) ◽  
pp. e02106-17 ◽  
Author(s):  
Miriam Becker ◽  
Lilo Greune ◽  
M. Alexander Schmidt ◽  
Mario Schelhaas
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Human Papillomaviruses ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Initial Infection ◽  
Structural Modifications ◽  
N Terminus ◽  
Rate Limiting

ABSTRACT Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid. IMPORTANCE HPVs are the main cause of anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicate that perhaps a unique niche for initial infection that could allow for rapid infection exists. In addition, our results may help to develop novel, preventive antiviral measures.

scholarly journals Reovirus core proteins λ1 and σ2 promote stability of disassembly intermediates and influence early replication events

2020 ◽  
Author(s):  
Stephanie Gummersheimer ◽  
Pranav Danthi
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Point Mutations ◽  
Host Cells ◽  
Capsid Proteins ◽  
Endosomal Membrane ◽  
The Core ◽  
Core Proteins ◽  
Early Replication ◽  
Outer Capsid

ABSTRACTThe capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of µ1-σ3 heterohexamers which surround the core. The core is comprised 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 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 has identified regions or specific residues within reovirus outer capsid 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 a lower ISVP stability and therefore converts to ISVP*s more readily. To identify the basis for lability of T3D/T1L L3S2, we screened for hyper-stable 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 from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection.IMPORTANCEProtein 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. 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 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.

scholarly journals Simian Virus 40 Assembly, Studied by Temperature-Induced Conformational Changes in Capsid Protein VP1

1986 ◽  
Vol 49 (1) ◽  
pp. 38-40 ◽  
Author(s):  
Minou Bina ◽  
Veronica Blasquez ◽  
Christine Ambrose
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Capsid Protein Vp1

scholarly journals A Monoclonal Antibody Specific for Reovirus Outer-Capsid Protein ς3 Inhibits ς1-Mediated Hemagglutination by Steric Hindrance

2001 ◽  
Vol 75 (14) ◽  
pp. 6625-6634 ◽  
Author(s):  
Emma L. Nason ◽  
J. Denise Wetzel ◽  
S. K. Mukherjee ◽  
Erik S. Barton ◽  
B. V. Venkataram Prasad ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Sialic Acid ◽  
Capsid Protein ◽  
Conformational Changes ◽  
Steric Hindrance ◽  
Host Cells ◽  
Capsid Proteins ◽  
Outer Capsid Protein ◽  
Viral Attachment ◽  
Outer Capsid

ABSTRACT Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein ς1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein ς3, blocks the binding of ς1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering ς1-ς3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 μg per ml, whereas that of Fabs was >200 μg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of ς3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins ς3 and μ1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.

scholarly journals Novel Structural Features of Human Norovirus Capsid

2019 ◽  
Author(s):  
Jessica Devant ◽  
Götz Hofhaus ◽  
Grant S. Hansman
Keyword(s):  
Structural Changes ◽  
Expression System ◽  
Structural Diversity ◽  
Structural Features ◽  
Amino Acid Identity ◽  
Icosahedral Symmetry ◽  
Capsid Shell ◽  
Capsid Protein Vp1 ◽  
Cell Expression ◽  
Insect Cell Expression

ABSTRACTHuman noroviruses are a major cause of gastroenteritis, yet there are still no vaccines or antivirals available. Nevertheless, a number of vaccine candidates that are currently in clinical trials are composed of norovirus virus-like particles (VLPs). These VLPs are recognized as morphologically and antigenically similar to norovirus virions. An X-ray crystal structure of the prototype (GI.1) VLPs showed that the norovirus capsid has a T=3 icosahedral symmetry and is composed of 180 copies of the major capsid protein (VP1) that folds into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM structure of VLPs for two GII.4 noroviruses that were detected in 1974 and 2012. We showed that these VLPs had a T=4 symmetry and were composed of 240 copies of VP1. The VP1 on the T=4 VLPs adapted four quasi-equivalent subunits (termed A, B, C, and D), which formed two distinct dimers (A/B and C/D). We found that the T=4 protruding domain was elevated ~21 Å off the capsid shell, which was ~7 Å more than the previously determined for the T=3 GII.10 norovirus. Another interesting feature of the T=4 VLPs was a small cavity and flaplike structure located at the twofold axis. This structural feature was associated with the shell domain (D subunit) and disrupted the contiguous shell. Altogether, we showed that the T=4 VLPs had a number of structural similarities and differences with other noroviruses, but how these structural changes associate with norovirus virions could be important for vaccine studies.IMPORTANCEThe discovery that the GII.4 VLPs (identified in 1974 and 2012, termed CHDC-1974 and NSW-2012, respectively) have a T=4 symmetry is of major significance, since the NSW-2012 is clinically important and previous structural and biochemical studies assumed noroviruses have a T=3 symmetry and are composed of 180 copies of VP1. More importantly, NSW-2012 norovirus shared 96% amino acid identity with a GII.4 vaccine candidate and our data suggests that this vaccine might also have a T=4 symmetry. Although it is not clear if the T=4 VLPs were an artifact of the insect cell expression system, the T=4 VLP vaccines might not recognize equivalent epitopes on T=3 virions, which will be important for future neutralization studies. Finally, further studies with other norovirus genotypes and virions are clearly needed in order to determine the level of this structural diversity.

scholarly journals Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment

2014 ◽  
Vol 89 (2) ◽  
pp. 1013-1023 ◽  
Author(s):  
Julian Scherer ◽  
Richard B. Vallee
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Motor Protein ◽  
Cytoplasmic Dynein ◽  
Low Ph ◽  
Virus Infectivity ◽  
Adenovirus Serotype ◽  
Serotype 5 ◽  
Ph Dependent

ABSTRACTVirus capsids provide genome protection from environmental challenges but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus serotype 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires the transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH but undergoes more subtle conformational changes. These changes are indicated by increased sensitivities to SDS-mediated dissociation and dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding by low-pH-treated hexon. Dispase cleavage, which we find maps to a specific site within hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry but completely inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and is strongly inhibited in the postentry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus.IMPORTANCEThe adenovirus serotype 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport toward the nucleus and AdV5 infectivity. In this work, we describe how low-pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1, which depends on the same structural changes and furthermore regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating long-known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

scholarly journals Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

2015 ◽  
Vol 89 (8) ◽  
pp. 4143-4157 ◽  
Author(s):  
Mike Strauss ◽  
David J. Filman ◽  
David M. Belnap ◽  
Naiqian Cheng ◽  
Roane T. Noel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Capsid Protein ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Cell Surface Receptor ◽  
Membrane Structures ◽  
Lipid Molecule ◽  
Surface Receptor ◽  
Capsid Protein Vp1

ABSTRACTPoliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called “pocket factor”), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid.IMPORTANCEThe cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.

scholarly journals Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Yong Luo ◽  
Nasim Motamedi ◽  
Thomas G. Magaldi ◽  
Gretchen V. Gee ◽  
Walter J. Atwood ◽  
...  
Keyword(s):  
Cell Surface ◽  
Capsid Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Host Cells ◽  
Wild Type ◽  
Vacuole Formation ◽  
Cytoplasmic Vacuolization ◽  
Capsid Protein Vp1 ◽  
Sv40 Infection

ABSTRACT Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40. IMPORTANCE The DNA tumor virus SV40 was discovered more than a half century ago as a contaminant of poliovirus vaccine stocks, because it caused dramatic cytoplasmic vacuolization of permissive host cells. Although SV40 played a historically important role in the development of molecular and cellular biology, restriction mapping, molecular cloning, and whole-genome sequencing, the basis of this vacuolization phenotype was unknown. Here, we show that SV40-induced vacuolization is triggered by the binding of the major viral capsid protein, VP1, to a cell surface ganglioside receptor, GM1. No other viral proteins or virus replication is required for vacuole formation. Other polyomaviruses utilize different ganglioside receptors, but they do not induce vacuolization. This work identifies the molecular trigger for the phenotype that led to the discovery of this important virus and provides the first molecular insight into an unusual and enigmatic cytopathic effect due to virus infection.

scholarly journals Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

2015 ◽  
Vol 89 (14) ◽  
pp. 7038-7052 ◽  
Author(s):  
Carla Cerqueira ◽  
Pilar Samperio Ventayol ◽  
Christian Vogeley ◽  
Mario Schelhaas
Keyword(s):  
Wound Healing ◽  
Serine Protease ◽  
Capsid Protein ◽  
Conformational Changes ◽  
Human Papillomaviruses ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Human Papillomavirus 16 ◽  
Surface Receptor

ABSTRACTThe entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved.IMPORTANCEOur analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures.

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

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Stephanie L. Gummersheimer ◽  
Pranav Danthi
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Point Mutations ◽  
Host Cells ◽  
Capsid Proteins ◽  
Endosomal Membrane ◽  
The Core ◽  
Core Proteins ◽  
Early Replication ◽  
Outer Capsid

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.

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