Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

P1 Glutamine isosteres in the design of inhibitors of 3C/3CL protease of human viruses of the Pisoniviricetes class.

Viral infections are one of the leading causes of acute morbidity in humans and much endeavour has been made by the synthetic community for the development of drugs to treat associated diseases. Peptide-based enzyme inhibitors, usually short sequences of three or four residues, are one of the classes of compounds currently under development for enhancement of their activity and pharmaceutical properties. This review reports the advances made in the design of inhibitors targeting the family of highly conserved viral proteases 3C/3CLpro, which play a key role in viral replication and present minimal homology with mammalian proteases. Particular focus is put on the reported development of P1 glutamine isosteres to generate potent inhibitors mimicking the natural substrate sequence at the site of recognition.'

Murine Norovirus: Additional Protocols for Basic and Antiviral Studies.

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents.

An Isomer of Galidesivir That Potently Inhibits Influenza Viruses and Members of the Bunyavirales Order.

We report for the first time the antiviral activities of two iminovirs (antiviral imino-C-nucleosides) 1 and 2, structurally related to galidesivir (Immucillin A, BCX4430). An iminovir containing the 4-aminopyrrolo[2,1-f][1,2,4-triazine] nucleobase found in remdesivir exhibited submicromolar inhibition of multiple strains of influenza A and B viruses, as well as members of the Bunyavirales order. We also report the first syntheses of ProTide prodrugs of iminovir monophosphates, which unexpectedly displayed poorer viral inhibition than their parent nucleosides in vitro. An efficient synthesis of the 4-aminopyrrolo[2,1-f][1,2,4-triazine]-containing iminovir 2 was developed to enable preliminary in vivo studies, wherein it displayed significant toxicity in BALB/c mice and limited protection against influenza. Further modification of this anti-influenza iminovir will therefore be required to improve its therapeutic value.

Human Norovirus Triggers Primary B Cell Immune Activation In Vitro.

Human norovirus (HNoV) is a global health and socioeconomic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3 days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next, we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+, memory-switched CD27+ IgD-, memory-unswitched CD27+ IgD+, and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar, changes in the B cell subset distribution upon infection were observed, which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly, primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro, which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover, HNoV does not elicit lifelong immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen, and lymph node specimens, while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses.

Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Protein Nucleotidylylation in +ssRNA Viruses.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

Norovirus VPg Binds RNA through a Conserved N-Terminal K/R Basic Patch.

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

Delivering Two Tumour Antigens Survivin and Mucin-1 on Virus-Like Particles Enhances Anti-Tumour Immune Responses.

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

Dry Formulation of Virus-Like Particles in Electrospun Nanofibers.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w/v) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Felis catus papillomavirus type 2 virus-like particle vaccine is safe and immunogenic but does not reduce FcaPV-2 viral loads in adult cats.

Felis catus papillomavirus type 2 (FcaPV-2) commonly infects the skin of domestic cats and has been associated with the development of skin cancer. In the present study, a FcaPV-2 virus-like particle (VLP) vaccine was produced and assessed for vaccine safety, immunogenicity, and impact on FcaPV-2 viral load. This is the first report of the use of a papillomavirus VLP vaccine in domestic cats. The FcaPV-2 VLP vaccine was given to ten adult cats that were naturally infected with FcaPV-2, and a further ten naturally infected cats were sham vaccinated as a control group. The rationale for vaccinating cats already infected with the virus was to induce neutralizing antibody titers that could prevent reinfection of new areas of skin and reduce the overall viral load, as has been demonstrated in other species. Reducing the overall FcaPV-2 viral load could reduce the risk for subsequent PV-associated cancer. The vaccine in this study was well-tolerated, as none of the cats developed any signs of local reaction or systemic illness. In the treatment group, the geometric mean anti-papillomavirus endpoint antibody titers increased significantly following vaccination from 606 (95% CI 192-1913) to 4223 (2023-8814), a 7.0-fold increase, although the individual antibody response varied depending on the level of pre-existing antibodies. Despite the immunogenicity of the vaccine, there was no significant change in FcaPV-2 viral load in the treatment group compared to the control group, over the 24 week follow-up period. A possible reason is that FcaPV-2 was already widespread in the basal skin layer of these adult cats and so preventing further cells from becoming infected had no impact on the overall viral load. Therefore, these results do not support the use of a FcaPV-2 VLP vaccine to reduce the risk for PV-associated cancer in cats in which FcaPV-2 infection is already well established. However, these results justify future studies in which the vaccine is administered to younger cats prior to FcaPV-2 infection becoming fully established.

Cell Cycle Arrest is a Conserved Function of Norovirus VPg Proteins.

Murine norovirus (MNV) viral protein genome-linked (VPg) manipulates the cell cycle to induce a G0/G1 arrest and gain a beneficial replication environment. All viruses of the norovirus genus encode a VPg protein; however, it is unknown if the G0/G1 arrest induced by MNV VPg is conserved in other members of the genus. RNA transcripts encoding a representative viral VPg from five norovirus genogroups were transfected into RAW-Blue murine macrophages, and the percentage of cells in each phase of the cell cycle was determined. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins tested, and in the wider Caliciviridae family the arrest was also conserved in rabbit hemorrhagic disease virus (RHDV) VPg and human sapovirus (HuSV) VPg. Truncation of MNV VPg shows that the first 62 amino acids are sufficient for a cell cycle arrest, and alignment of VPg sequences revealed a conserved motif in the N-terminal region of VPg. Analysis of VPg constructs with single N-terminal region point mutations, or exchange of N-terminal regions between VPg proteins, confirmed the importance of the N-terminal region for cell cycle arrest. These results provide evidence that G0/G1 cell cycle arrest is a conserved function of norovirus VPg proteins that involves the N-terminal region of these proteins.

Functionalisation of Virus-Like Particles Enhances Antitumour Immune Responses.

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

Virus-like particle vaccines: immunology and formulation for clinical translation.

INTRODUCTION: Virus-like particle (VLP) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. While some VLP vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. Often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a VLP vaccine, despite any preclinical advantages identified over the competition. Novelty, adaptability and formulation compatibility may prove invaluable in helping place VLP vaccines at the forefront of vaccination technology. AREAS COVERED: The purpose of this review is to outline the diversity of VLP vaccines, VLP-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of VLP vaccines. EXPERT COMMENTARY: The role of formation science, with an emphasis on the diversity of immune responses induced by VLP, is underrepresented amongst clinical trials for VLP vaccines. Harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of VLP vaccines.

Multi-target chimaeric VLP as a therapeutic vaccine in a model of colorectal cancer.

BACKGROUND: Colorectal cancer is responsible for almost 700,000 deaths annually worldwide. Therapeutic vaccination is a promising alternative to conventional treatment for colorectal cancer, using vaccines to induce targeted immune responses against tumour-associated antigens. In this study, we have developed chimaeric virus-like particles (VLP), a form of non-infectious non-replicative subunit vaccine consisting of rabbit haemorrhagic disease virus (RHDV) VP60 capsid proteins containing recombinantly inserted epitopes from murine topoisomerase IIα and survivin. These vaccines were developed in mono- (T.VP60, S.VP60) and multi-target (TS.VP60) forms, aiming to elucidate the potential benefits from multi-target vaccination. METHODS: Chimaeric RHDV VLP were developed by recombinantly inserting immune epitopes at the N-terminus of VP60. Vaccines were tested against a murine model of colorectal cancer by establishing MC38-OVA tumours subcutaneously. Unmethylated CpG DNA oligonucleotides (CpGs) were used as a vaccine adjuvant. Statistical tests employed included the Mantel-Cox log-rank test, ANOVA and unpaired t-tests depending on the data analysed, with a post hoc Bonferroni adjustment for multiple measures. RESULTS: Chimaeric RHDV VLP were found to form a composite particle in the presence of CpGs. Overall survival was significantly improved amongst mice bearing MC38-OVA tumours following vaccination with T.VP60 (60%, 9/15), S.VP60 (60%, 9/15) or TS.VP60 (73%, 11/15). TS.VP60 significantly prolonged the vaccine-induced remission period in comparison to each mono-therapy. CONCLUSIONS: Chimaeric VLP containing multiple epitopes were found to confer an advantage for therapeutic vaccination in a model of colorectal cancer based on the prolongation of remission prior to tumour escape.

Benign Rabbit Calicivirus in New Zealand.

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand.IMPORTANCE Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols.

Transcriptomic analysis of human norovirus NS1-2 protein highlights a multifunctional role in murine monocytes.

BACKGROUND: The GII.4 Sydney 2012 strain of human norovirus (HuNoV) is a pandemic strain that is responsible for the majority of norovirus outbreaks in healthcare settings. The function of the non-structural (NS)1-2 protein from HuNoV is unknown. RESULTS: In silico analysis of human norovirus NS1-2 protein showed that it shares features with the murine NS1-2 protein, including a disordered region, a transmembrane domain and H-box and NC sequence motifs. The proteins also contain caspase cleavage and phosphorylation sites, indicating that processing and phosphorylation may be a conserved feature of norovirus NS1-2 proteins. In this study, RNA transcripts of human and murine norovirus full-length and the disordered region of NS1-2 were transfected into monocytes, and next generation sequencing was used to analyse the transcriptomic profile of cells expressing virus proteins. The profiles were then compared to the transcriptomic profile of MNV-infected cells. CONCLUSIONS: RNAseq analysis showed that NS1-2 proteins from human and murine noroviruses affect multiple immune systems (chemokine, cytokine, and Toll-like receptor signaling) and intracellular pathways (NFκB, MAPK, PI3K-Akt signaling) in murine monocytes. Comparison to the transcriptomic profile of MNV-infected cells indicated the pathways that NS1-2 may affect during norovirus infection.

Expression of the NS5 (VPg) Protein of Murine Norovirus Induces a G1/S Phase Arrest.

Murine norovirus-1 (MNV-1) is known to subvert host cell division inducing an accumulation of cells in the G0/G1 phase, creating conditions where viral replication is favored. This study identified that NS5 (VPg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to MNV-1 infection. NS5 expression induced an accumulation of cells in the G0/G1 phase in an asynchronous population by inhibiting progression at the G1/S restriction point. Furthermore, NS5 expression resulted in a down-regulation of cyclin A expression in asynchronous cells and inhibited cyclin A expression in cells progressing from G1 to S phase. The activity of NS5 on the host cell cycle occurs through an uncharacterized function. Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G0/G1 phase as identified for wild-type NS5. To the best of our knowledge, this is the first report of a VPg protein manipulating the host cell cycle.

Benign Rabbit Caliciviruses Exhibit Evolutionary Dynamics Similar to Those of Their Virulent Relatives.

UNLABELLED: Two closely related caliciviruses cocirculate in Australia: rabbit hemorrhagic disease virus (RHDV) and rabbit calicivirus Australia 1 (RCV-A1). RCV-A1 causes benign enteric infections in the European rabbit (Oryctolagus cuniculus) in Australia and New Zealand, while its close relative RHDV causes a highly pathogenic infection of the liver in the same host. The comparison of these viruses provides important information on the nature and trajectory of virulence evolution, particularly as highly virulent strains of RHDV may have evolved from nonpathogenic ancestors such as RCV-A1. To determine the evolution of RCV-A1 we sequenced the full-length genomes of 44 RCV-A1 samples isolated from healthy rabbits and compared key evolutionary parameters to those of its virulent relative, RHDV. Despite their marked differences in pathogenicity and tissue tropism, RCV-A1 and RHDV have evolved in a very similar manner. Both viruses have evolved at broadly similar rates, suggesting that their dynamics are largely shaped by high background mutation rates, and both exhibit occasional recombination and an evolutionary environment dominated by purifying selection. In addition, our comparative analysis revealed that there have been multiple changes in both virulence and tissue tropism in the evolutionary history of these and related viruses. Finally, these new genomic data suggest that either RCV-A1 was introduced into Australia after the introduction of myxoma virus as a biocontrol agent in 1950 or there was drastic reduction of the rabbit population, and hence of RCV-A1 genetic diversity, perhaps coincident with the emergence of myxoma virus. IMPORTANCE: The comparison of closely related viruses that differ profoundly in propensity to cause disease in their hosts offers a powerful opportunity to reveal the causes of changes in virulence and to study how such changes alter the evolutionary dynamics of these pathogens. Here we describe such a novel comparison involving two closely related RNA viruses that cocirculate in Australia, the highly virulent rabbit hemorrhagic disease virus (RHDV) and the nonpathogenic rabbit calicivirus Australia 1 (RCV-A1). Both viruses infect the European rabbit, but they differ in virulence, tissue tropism, and mechanisms of transmission. Surprisingly, and despite these fundamental differences, RCV-A1 and RHDV have evolved at very similar (high) rates and with strong purifying selection. Furthermore, candidate key mutations were identified that may play a role in virulence and/or tissue tropism and therefore warrant further investigation.

Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12.

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.