Biology subjects

Philip, A. A.

Publications and source records attributed to Philip, A. A..

7 recordsLinked to original sources

bioRxiv2024

Viroporin activity from rotavirus nonstructural protein 4 induces intercellular calcium waves that contribute to pathogenesis

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro, organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4s ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

microbiology↗

bioRxiv2023

Recombinant Rotavirus Expressing the Glycosylated S1 Protein of SARS-CoV-2

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment 7 (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment 7 RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the [~]120-kDa S1 protein. The [~]120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of [~]80 kDa. Co-pulldown assays demonstrated that the [~]120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibody, suggesting the rSA11/S1f virus expressed an authentic form of S1. Probably due to the presence of FLAG tags on their S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

microbiology↗

bioRxiv2022

Rotavirus NSP1 subverts the antiviral OAS-RNase L pathway by inducing RNase L degradation

The interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS) - RNase L pathway plays a critical role in antiviral immunity. Group A rotaviruses, including the simian SA11 strain, inhibit this pathway through two activities: an E3-ligase related activity of NSP1 that degrades proteins necessary for IFN signaling, and a phosphodiesterase (PDE) activity of VP3 that hydrolyzes the RNase L-activator 2',5'-oligoadenylate. Unexpectedly, we found that a recombinant (r) SA11 double-mutant virus deficient in both activities (rSA11-VP3H797R-NSP1{Delta}C17) retained the ability to prevent RNase L activation. Mass spectrometry led to the discovery that NSP1 interacts with RNase L in rSA11-infected MA104 cells. This interaction was confirmed through co-pulldown assay of cells transiently expressing NSP1 and RNase L. Immunoblot analysis showed that infection with wild type rSA11 virus, rSA11-VP3H797R-NSP1{Delta}C17 double-mutant virus, or single mutant forms of the latter virus, all resulted in the depletion of endogenous RNase L. The loss of RNase L was reversed by addition of the neddylation inhibitor MLN4924, but not the proteasome inhibitor MG132. Analysis of additional mutant forms of rSA11 showed that RNase L degradation no longer occurred when either the N-terminal RING domain of NSP1 was mutated or the C-terminal 98 amino acids of NSP1 were deleted. The C-terminal RNase L degradation domain is positioned upstream and is functionally independent of the NSP1 domain necessary for inhibiting IFN expression. Our studies reveal a new role for NSP1 and its E3-ligase related activity as an antagonist of RNase L and uncover a novel virus-mediated strategy of inhibiting the OAS-RNase L pathway. IMPORTANCEFor productive infection, rotavirus and other RNA viruses must suppress interferon (IFN) signaling and the expression of IFN-stimulated antiviral gene products. Particularly important is inhibiting the interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS) - RNase L pathway, as activated RNase L can direct the nonspecific degradation of viral and cellular RNAs, thereby blocking viral replication and triggering cell death pathways. In this study, we have discovered that the simian SA11 strain of rotavirus employs a novel strategy of inhibiting the OAS-RNase L pathway. This strategy is mediated by SA11 NSP1, a nonstructural protein that hijacks E3 cullin-RING ligases, causing the ubiquitination and degradation of host proteins essential for IFN induction. Our analysis shows that SA11 NSP1 also recognizes and causes the ubiquitination of RNase L, an activity resulting in depletion of endogenous RNase L. These data raise the possibility of using therapeutics targeting cellular E3 ligases to control rotavirus infections.

microbiology↗

bioRxiv2022

Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins

Rotaviruses, segmented dsRNA viruses of the Reoviridae family, are a primary cause of acute gastroenteritis in young children. In countries where rotavirus vaccines are widely used, norovirus (NoV) has emerged as the major cause of acute gastroenteritis. Towards the goal of creating a combined rotavirus-NoV vaccine, we explored the possibility of generating recombinant rotaviruses (rRVs) expressing all or portions of NoV GII.4 VP1 capsid proteins. This was accomplished by replacing the segment 7 NSP3 ORF with a cassette encoding sequentially NSP3, a 2A stop-restart translation element, and all or portions (P, P2) of NoV VP1. In addition to successfully recovering SA11 rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the RIX4414 segment 7 RNA. Immunoblot assay confirmed that rRVs expressed NoV capsid proteins as independent products. Moreover, VP1 expressed by rRVs underwent dimerization and was recognized by conformational-dependent anti-VP1 antibodies. Serially passaged rRVs that expressed the NoV P and P2 were genetically stable, retaining sequences up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.5 kb VP1 sequence were less stable and gave rise to virus populations with segment 7 RNAs lacking VP1 coding sequences. Together, these studies suggest that it may be possible to develop combined rotavirus-NoV vaccines using modified segment 7 RNA to express NoV P or P2. In contrast, development of potential rotavirus-NoV vaccines expressing NoV VP1 will need additional efforts to improve genetic stability. ImportanceRotavirus (RV) and norovirus (NoV) are the two most important causes of viral acute gastroenteritis (AGE) in young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of live attenuated RV vaccines, similar highly effective NoV vaccines are not available. To pursue the development of a combined RV-NoV vaccine, we examined the potential usefulness of RV as an expression vector of all or portions of the NoV capsid protein VP1. Our results showed that by replacing the NSP3 open reading frame in RV genome segment 7 RNA with a coding cassette for NSP3, a 2A stop-restart translation element, and VP1, recombinant RVs can be generated that express NoV capsid proteins as separate products. These findings raise the possibility of developing a new generation of RV-based combination vaccines that can provide protection against a second enteric pathogen, such as the NoV.

microbiology↗

bioRxiv2021

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment. ImportanceAmong the vaccines administered to children in the US and many other countries are those targeting rotavirus, a segmented double-stranded RNA virus that is a major cause of severe gastroenteritis. In this study, we have examined the feasibility of modifying the rotavirus genome by reverse genetics, such that the virus could serve as an expression vector of the SARS-CoV-2 spike protein. Results were obtained showing that recombinant rotaviruses can be generated that express domains of the SARS CoV-2 spike protein, including the receptor-binding domain (RBD), a common target of neutralizing antibodies produced in individuals infected by the virus. Our findings raise the possibility of creating a combined rotavirus-COVID-19 vaccine that could be used in place of current rotavirus vaccines.

microbiology↗

bioRxiv2020

Expression of Separate Foreign Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element

The segmented 18.5-kB dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently-developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate foreign protein, in addition to the 12 viral proteins. To address this, we replaced the NSP3 open-reading-frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus 2A-like self-cleaving element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing, generally genetically stable, and expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 foreign proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kB and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple foreign proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses may prove useful as expression vectors. ImportanceRotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate foreign protein, in addition to the full complement of viral proteins. Through this approach, we have generated wildtype-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live-cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of foreign RNA sequence, raising the possibility of using the virus as vaccine expression vector.

microbiology↗

bioRxiv2019

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic Ca2+ signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced Ca2+ signaling.

microbiology↗