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. NSP4's 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.

Recovery of Recombinant Rotaviruses by Reverse Genetics.

Rotaviruses are the primary cause of severe gastroenteritis in infants and young children throughout the world. To combat rotavirus illness, several live oral vaccines have been developed, or are under development, that are formulated from attenuated human or human-animal reassortant strains of rotavirus. While the effectiveness of these vaccines is generally high in developed countries, the same vaccines are significantly less effective in many developing countries, where the need for rotavirus vaccines is greatest. Recently, reverse genetics systems have been developed that allow modification of the segmented double-stranded (ds)RNA genome of rotavirus, including reprogramming the genome to allow expression of additional proteins that may stimulate expanded neutralizing antibody responses in vaccinated children. The use of reverse genetics systems may not only lead to the development of more potent classes of vaccines but can be used to better explore the intricacies of rotavirus molecular biology and pathogenesis. In this article, we share protocols that can be used to generate recombinant rotaviruses, including modified strains that express foreign proteins.

T7 expression plasmids for producing a recombinant human G1P[8] rotavirus comprising RIX4414 sequences of the RV1 (Rotarix , GSK) vaccine strain.

The live oral rotavirus RV1 (Rotarix) vaccine is formulated from the human G1P[8] RIX4414 virus. Based on RIX4414 sequences, T7 expression plasmids were constructed that supported recovery of recombinant RIX4414-like viruses by reverse genetics. These plasmids will advance the study of the RV1 vaccine, possibly allowing improvements to its efficacy.

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 seven (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 seven 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 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the 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.

Rotavirus NSP1 Subverts the Antiviral Oligoadenylate Synthetase-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-NSP1ΔC17) retained the ability to prevent RNase L activation. Mass spectrometry led to the discovery that NSP1 interacts with RNase L in rSA11-infected HT29 cells. This interaction was confirmed through copulldown assay of cells transiently expressing NSP1 and RNase L. Immunoblot analysis showed that infection with wild-type rSA11 virus, rSA11-VP3H797R-NSP1ΔC17 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. IMPORTANCE For 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.

Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins.

Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, is 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 the NoV GII.4 VP1 capsid protein. This was accomplished by replacing the segment 7 NSP3 open reading frame 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 rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the segment 7 RNA of the RIX4414 vaccine strain. An 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 additional sequences of up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.6-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. IMPORTANCE Rotavirus (RV) and norovirus (NoV) are the two most important causes of acute viral gastroenteritis (AGE) in infants and young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of universal mass vaccination with 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 of using 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. These findings raise the possibility of developing new generations of RV-based combination vaccines that provide protection against a second enteric pathogen, such as NoV.

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

Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (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 immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.

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.

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

The segmented 18.5-kbp 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 heterologous 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 translational 2A stop-restart 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 and generally genetically stable, and they 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 heterologous 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 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous 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 will prove useful as expression vectors.IMPORTANCE Rotaviruses 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 heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-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 heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System.

Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

Collection of Recombinant Rotaviruses Expressing Fluorescent Reporter Proteins.

A collection of recombinant rotaviruses that express the fluorescent markers UnaG, mKate, mRuby, TagBFP, CFP, or YFP as separate proteins was generated. Genes for the fluorescent proteins were inserted into genome segment 7 without compromising expression of the protein NSP3. These recombinant rotaviruses are valuable for analyzing rotavirus biology by fluorescence-based live-cell imaging.

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 calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.

An interrater and intrarater reliability study of 3 photographic scales for the classification of perioral aesthetic features.

BACKGROUND: Validated aesthetic rating scales for the perioral area provide objective evaluations for clinical trials and practice. OBJECTIVE: To confirm the reliability of 3 scales for evaluating dermal filler and neurotoxin treatments of the perioral area. MATERIALS AND METHODS: Three lip-specific photographic scales were developed from standardized 2-dimensional images to evaluate Perioral Lines at Rest (POL), Oral Commissures (OCS), and Perioral Lines at Maximum Contraction (POLM) severity scales. Each 4-grade scale (none to severe) had 3 representative images per grade. Physician validators rated volunteers on each scale (2 rounds of live review). Volunteers provided 2 series of self-assessments. Physician and subject intrarater reliability were based on the comparison of round 1 and round 2 scores (mean weighted kappa coefficient). Other measures were physician interrater agreement (intraclass correlation) and subject/physician interrater agreement (Pearson correlation). RESULTS: Physician intrarater agreement was almost perfect or substantial (POL, 0.725; OCS, 0.789; POLM, 0.826). Overall, physician interrater agreement was almost perfect for all 3 scales and ranged from moderate to substantial by grade. Subject intrarater agreement and subject/physician interrater agreement were substantial. CONCLUSION: All scales demonstrated a high degree of intrarater and interrater reliability during the validation process. Physician concordance was good; subject ratings were reliable and comparable to physician assessments.

Long-term treatment of glabellar rhytides using onabotulinumtoxina.

OBJECTIVE: To determine whether injection every 4 months with onabotulinumtoxinA provides longer-lasting reduction or elimination of glabellar rhytides. METHODS: Toxin-naive women (N=45) aged 30 to 50 with moderate to severe glabellar lines at maximum contraction completed the study. Five sites were treated with 4 U of onabotulinumtoxinA. Participants were assessed at day 30; those with glabellar severity of none to mild remained in the study. Participants received identical treatments at months 4, 8, 12, 16, and 20, and were assessed without treatment at 24, 25, and 26 months. RESULTS: Baseline physician- and participant-rated treatment responders at maximum contraction were 84% and 80%, respectively, at month 20; and 56% and 58%, respectively, at month 26. Mean changes from physician- and participant-assessed Facial Wrinkle Scale at maximum contraction from baseline to all postbaseline measurements were significant (p≤.001). The percentage of participants reporting neutral (0) to couldn't be more satisfied (+4) with their appearance ranged from 91% to 100% during months 1 to 24. At month 26, 87% of participants reported satisfaction, with appearance scores between 0 and +4. No adverse events were reported. CONCLUSIONS: OnabotulinumtoxinA delivered for 20 months significantly reduces or progressively eliminates glabellar rhytides for up to 6 months after the last treatment.