Mariam B. Gonzalez-Hernandez

Enteric bacteria promote human and mouse norovirus infection of B cells

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science, this issue p. 755; see also p. 700 Gut bacteria that express histo-blood group antigens help human norovirus to infect B cells. [Also see Perspective by Robinson and Pfeiffer] The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual’s histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

Efficient Norovirus and Reovirus Replication in the Mouse Intestine Requires Microfold (M) Cells

ABSTRACT Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. M cells have also been suggested as a portal for pathogen entry into the host. While virus particles have been observed in M cells, it is not known whether viruses use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect host organisms via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells and provides a tractable experimental system for understanding how an enteric virus overcomes the intestinal epithelial barrier to infect underlying target cells. We found that replication of two divergent MNV strains was reduced in mice depleted of M cells. Reoviruses are double-stranded RNA viruses that infect hosts via respiratory or enteric routes. In contrast to MNV, reovirus infects enterocytes in the intestine. Despite differences in cell tropism, reovirus infection was also reduced in M cell-depleted mice. These data demonstrate that M cells are required for the pathogenesis of two unrelated enteric viruses that replicate in different cell types within the intestine. IMPORTANCE To successfully infect their hosts, pathogens that infect via the gastrointestinal tract must overcome the multilayered system of host defenses. Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. Virus particles have been observed within M cells. However, it is not known whether viruses use M cells to initiate a productive infection. To address this question, we use MNV and reovirus, two enteric viruses that replicate in different cell types in the intestine, intestinal epithelial cells for reovirus and intestinal mononuclear phagocytes for MNV. Interestingly, MNV- and reovirus-infected mice depleted of M cells showed reduced viral loads in the intestine. Thus, our work demonstrates the importance of M cells in the pathogenesis of enteric viruses irrespective of the target cell type in which the virus replicates.

Plaque assay for murine norovirus.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses

ABSTRACT Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

Oral Norovirus Infection Is Blocked in Mice Lacking Peyer's Patches and Mature M Cells.

ABSTRACT A critical early step in murine norovirus (MNV) pathogenesis is crossing the intestinal epithelial barrier to reach the target cells for replication, i.e., macrophages, dendritic cells, and B cells. Our previous work showed that MNV replication decreases in the intestines of mice conditionally depleted of microfold (M) cells. To define the importance of Peyers patch (PP) M cells during MNV pathogenesis, we used a model of BALB/c mice deficient in recombination-activating gene 2 (Rag2) and the common gamma chain (γc) (Rag-γc−/−), which lack gut-associated lymphoid tissues (GALT), such as Peyers patches, and mature GP2+ M cells. Rag-γc−/− mice were infected intraperitoneally or perorally with MNV-1 or CR3 for 24 or 72 h. Although the intestinal laminae propriae of Rag-γc−/− mice have a higher frequency of certain MNV target cells (dendritic cells and macrophages) than those of wild-type mice and lack others (B cells), Rag-γc−/− and wild-type BALB/c mice showed relatively similar viral loads in the intestine following infection by the intraperitoneal route, which provides direct access to target cells. However, Rag-γc−/− mice were not productively infected with MNV by the oral route, in which virions must cross the intestinal epithelial barrier. These data are consistent with a model whereby PP M cells are the primary route by which MNV crosses the intestinal epithelia of BALB/c mice. IMPORTANCE Noroviruses (NoVs) are prevalent pathogens that infect their hosts via the intestine. Identifying key factors during the initial stages of virus infection in the host may provide novel points of intervention. Microfold (M) cells, antigen-sampling cells in the intestine, were previously shown to provide a gateway for murine NoV (MNV) into the host, but the relative importance of this uptake pathway remained unknown. Here we show that the absence of gut-associated lymphoid tissues (GALT), such as Peyers patches, which contain high numbers of mature M cells, renders BALB/c mice refractory to oral infection with MNV. These findings are consistent with the model that M cells represent the primary route by which MNV crosses the intestinal epithelial barrier and infects underlying immune cells during a productive infection.

Multiple effects of dendritic cell depletion on murine norovirus infection

Dendritic cells (DCs) are permissive to murine norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DCs during infection, conventional DCs were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titres in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analysed later during infection. Depletion of conventional DCs resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DCs play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DCs in controlling and/or facilitating a norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DCs control MNV replication and promote viral dissemination.

Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells

Accurate cell‐size determinations support the prediction that serum starvation and related whole‐culture methods cannot synchronize cells. Theoretical considerations predict that whole‐culture methods of synchronization cannot synchronize cells. Upon serum starvation, the fraction of cells with a G1‐phase amount of DNA increased, but the cell‐size distribution is not narrowed. In true synchronization, the cell‐size distribution should be narrower than the cell‐size distribution of the original culture. In contrast, cells produced by a selective (i.e. non‐whole‐culture) method have a specific DNA content, a narrow size distribution, and divide synchronously. The general theory leading to the conclusion that whole‐culture methods for synchronization do not work implies that one can generalize these serum‐starvation results to other cell lines and other whole‐culture methods, to conclude that these methods do not synchronize cells.

Membrane-elution analysis of content of cyclins A, B1, and E during the unperturbed mammalian cell cycle

BackgroundProblems with whole-culture synchronization methods for the study of the cell cycle have led to the need for an analysis of protein content during the cell cycle of cells that have not been starved or inhibited. The membrane-elution method is a method that allows the study of the cell cycle by producing a culture of unperturbed, synchronized cells.ResultsThe Helmstetter membrane-elution method for the continuous production of newborn, unperturbed, mammalian cells has been enhanced so that the collection of cells of different cell cycle ages is automated, reproducible, and relatively inexpensive. We have applied the automated membrane-elution method to the analysis of cyclin content during the cell cycle. Cyclin E protein was invariant during the cell cycle. Cyclins B1 and A accumulated continuously during the cell cycle and were degraded at mitosis. Newborn cells had ~0.5% of the cyclin B1 content of dividing cells.ConclusionThe expression patterns of cyclins A, B1, and E can be explained by constant mRNA levels during the cell cycle. Previously reported phase specific variations of the cyclins are not strictly necessary for cell-cycle progression. Cells produced by membrane-elution are available to other laboratories for analysis of the cell cycle.

Natural Secretory Immunoglobulins Enhance Norovirus Infection

Secretory immunoglobulins (SIg) are a first line of mucosal defense by the host. They are secreted into the gut lumen via the polymeric immunoglobulin receptor (pIgR) where they bind to antigen and are transported back across the FAE via M cells. Noroviruses are highly prevalent, enteric pathogens that cause significant morbidity, mortality and economic losses worldwide. Murine norovirus (MNV) exploits microfold (M) cells to cross the lymphoid follicle-associated epithelium (FAE) and infect the underlying population of immune cells. However, whether natural, innate SIg can protect against norovirus infection remains unknown. To investigate the role of natural SIg during murine norovirus pathogenesis, we used pIgR-deficient animals, which lack SIg in the intestinal lumen. Contrary to other enteric pathogens, acute MNV replication was significantly reduced in the gastrointestinal tract of pIgR-deficient animals compared to controls, despite increased numbers of dendritic cells, macrophages, and B cells in the Peyer’s patch, established MNV target cell types. Also, natural SIg did not alter MNV FAE binding or FAE crossing into the lymphoid follicle. Instead, further analysis revealed enhanced baseline levels of the antiviral molecules interferon gamma (IFNɣ) and inducible nitric oxide synthase (iNOS) in the small intestine of naive pIgR-deficient animals compared to controls. Removing the microbiota equalized IFNɣ and iNOS transcript levels as well as MNV viral loads in germ-free pIgR KO mice compared to germ-free controls. These data are consistent with a model whereby SIg sensing reduces pro-inflammatory, antiviral molecules, which facilitates intestinal homeostasis but thereby promotes MNV infection. In conclusion, these findings demonstrate that natural SIg are not protective during norovirus infection in mice and represent another example of indirect modulation of enteric virus pathogenesis by the microbiota.