High-Resolution Cryo-Electron Microscopy Structures of Murine Norovirus 1 and Rabbit Hemorrhagic Disease Virus Reveal Marked Flexibility in the Receptor Binding Domains

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to ∼8-Å resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.Murine norovirus 1 (MNV-1) (3, 14, 15) and rabbit hemorrhagic disease virus (RHDV) are members of the genera Norovirus and Lagovirus of the family Caliciviridae that offer a comparison to recombinant human norovirus (rNV) virus-like particles (VLPs) for assessing the structures and roles of domains within the capsid proteins of this family of viruses. Calicivirus particles contain 180 copies of the 56- to 76-kDa major capsid protein (Orf2), which is comprised of the internal/buried N terminus (N), shell (S), and protruding (P) domains (9, 10). The S domain, an eight-stranded β-barrel, forms an ∼300-Å contiguous shell around the RNA genome. A flexible hinge connects the shell to a “protruding” (P) domain at the C-terminal half of the capsid protein, which can be further divided into a globular head region (P2) and a stem region (P1) that connects the shell domain to P2. The accompanying article (13) describes the determination of the structure of the P domain of MNV-1 to a resolution of 2.0 Å.We recently determined the cryo-transmission electron microscopy (TEM) structure of MNV-1 to ∼12-Å resolution (4) and found that, compared to rNV VLPs (10) and San Miguel sea lion virus (SMSV) (1, 2), the protruding domains are rotated by ∼40° in a clockwise fashion and lifted up by ∼16 Å. To better understand the unusual conformation of MNV-1 and whether it is unique to this particular member of the calicivirus family, the ∼8-Å cryo-TEM structures of infectious MNV-1 and the VLPs of RHDV were determined.MNV-1 was produced as previously described (4). Three liters of cell culture yielded 0.5 to 1.0 mg of purified virus with a particle/PFU ratio of less than 100. Baculovirus expression and purification of recombinant RHDV (rRHDV) VLPs were performed as previously described (8). Cryo-electron microscopy (EM) data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) facility in San Diego, CA (4). Images were collected at a nominal magnification of ×50,000 at a pixel size of 0.1547 nm at the specimen level using Leginon software (12) and processed with Appion software (5). The contrast transfer function for each set of particles from each image was estimated and corrected using ACE2 (a variation of ACE [7]). Particle images were automatically selected (11). The final stacks of particle images contained 20,425 MNV virions and 7,856 rRHDV VLPs, and EMAN 3D (6) was used for the reconstructions. Resolutions were estimated by Fourier shell correlations (FSC) of the three-dimensional (3D) reconstructions and application of a cutoff of 0.5. An amplitude correction of the final electron density was performed using GroEL small-angle X-ray scattering (SAXS) data.3D reconstructions of MNV-1 and rRHDV were calculated to resolutions of 8 Å and 8.1 Å, respectively (Fig. ​(Fig.1).1). The P domains of rNV VLPs rest directly on top of the shell domain (10) (Fig. ​(Fig.1A).1A). In contrast, the P domains of MNV-1 are lifted and rotated above the shell of the capsid (4) (Fig. ​(Fig.1B).1B). At this higher resolution, there was a clear connection between the P1 domain and the shell domain in all three capsid subunits (Fig. ​(Fig.1B,1B, arrow A). Unlike the smooth protruding domains of rNV, MNV-1 has two clear “horns” (arrow B), not dissimilar to those observed for the sapoviruses (1, 2). There also are islands of density in the interior of the shell, directly beneath the 5-fold axes, that may represent ordered regions of RNA.Open in a separate windowFIG. 1.Stereo diagrams (left) and thin sections (right), with radius coloring, of rNV (A), MNV-1 (B), and an rRHDV VLP (C). For rNV, the atomic coordinates (10) were used. In MNV, arrow A indicates the thin connector between the P1 and S domains. Arrow B denotes the horns found at the tips of the P2 domains. Arrow C denotes the large gap between the P1 and S domains in the rRHDV VLP. Arrow D denotes the false connectivity in rRHDV VLPs between the P1 domain and the S domain near the 5-fold axes.As with MNV-1, there is a marked gap between the P and S domains in the rRHDV VLP (Fig. ​(Fig.1C,1C, arrow C). This gap is not as pronounced as in MNV-1 because the P domains are not rotated as in MNV-1. In this electron density map, the A/B dimers appear to be touching the shell domain near the 5-fold axes. This contact difference between the A/B dimers and the C/C dimers could be the reason why the tops of the C/C dimers appear to be markedly disordered compared to the A/B dimers in rRHDV and the C/C dimers in MNV-1.Shown in Fig. ​Fig.22 is the fitting of the atomic structures of the MNV-1 P domain (13) and the rNV S domains into the MNV-1 3D reconstruction electron density. The horns (arrow A, loops A′-B′ and E′-F′) observed at the tips of the P domain match exceedingly well with the electron density. As discussed in the accompanying publication (13), the A′-B′ and E′-F′ loops displayed two discrete conformations, a closed structure, where the two loops were tightly associated, and an open structure, where the loops were splayed apart. The horns of the closed conformation fit better into the reconstruction, as the E′-F′ loop in the open form jutted out of the density at the base of the horns. The unmodified density in the lower panel of Fig. ​Fig.22 shows fine features in the shell domain and a very clear connection between the shell and P1 domains. The connections between the P1 and S domains were of sufficient quality to build a basic backbone model by uncoiling the linker region (arrow B). The P domain in the unfiltered 3D reconstruction was far less ordered than the S domain (Fig. ​(Fig.2).2). This was likely due to movement of the entire P domain with respect to the shell.Open in a separate windowFIG. 2.Fitting of the MNV-1 P domain and the rNV shell domain into the MNV-1 electron density. A, B, and C subunits are represented by blue, green, and red, respectively. The electron density is shown in transparent gray. The top panel is the 8.0-Å-resolution 3D reconstruction modified using a low-pass filter. The bottom panel is the reconstruction without modification. The horns on the tops of the P domains are denoted by arrow A. Arrow B denotes the connection between the S and P domains.Using the structure of rNV VLP P domains for modeling, the rRHDV P domains are lifted off the surface of the shell, but not rotated as with MNV-1. This places the bottom edge of the A subunit P1 domain near the S domain at the 5-fold axes. The P-domain dimers of rNV and rRHDV have a more “arch-like” shape than MNV-1. Unlike in MNV-1, the electron densities of the C/C dimers in rRHDV are far more diffuse than those of the A/B dimers (Fig. ​(Fig.3B)3B) and the connector between the S and P1 domains is not clear. During fitting, the connector region was not as extended as with MNV-1. This may afford greater flexibility, leading to more diffuse electron density.Open in a separate windowFIG. 3.Fitting of the rNV atomic structure into the rRHDV VLP electron density. The upper stereo image shows the 8.1-Å-resolution 3D reconstruction after modification by a low-pass filter. Below is the same reconstruction prior to density modification.When the atomic models for the MNV-1 P domains (13) were placed into the cryo-TEM electron density (Fig. ​(Fig.4),4), the C termini extended deep into the cores of adjacent P domains. Possible connections not accounted for by the P-domain structures were also observed in the electron density between the P domains. A bulge between the P1 and P2 domains in the 3D reconstruction indicated a possible interaction between the C termini and the adjacent P domains. These same interactions were observed in the crystal lattice. This highly mobile C terminus may be a flexible tether between the P domains in the intact virion.Open in a separate windowFIG. 4.Possible carboxyl-terminus interactions between the P domains of MNV-1. (A) Stereo image of MNV-1 calculated to 12-Å resolution with (red) and without (yellow) the last 10 residues of the P domain. (B) The calculated MNV-1 density with the carboxyl terminus removed (yellow) overlaid onto the 3D reconstruction of MNV-1 (blue). Note the strands of difference density that roughly correspond to the C terminus in panel A. (C) The C-terminus interactions observed in the structure of the MNV-1 P domains. Shown in blue and green are ribbon diagrams of an A/B P-domain dimer. In mauve is a surface rendering of the C terminus from a crystallographically related dimer. (D) Surface rendering of the final MNV-1 model with possible interactions between the P domains in MNV-1. The carboxyl termini of the A subunits (blue) interact with the counterclockwise-related B subunits around the 5-fold axes (white arrows). Around the 3-fold (quasi-6-fold) axes, the C subunits interact with the A subunits and the B subunits interact with the C subunits (orange arrows).It is absolutely clear that the hinge region between the S and P domains affords a remarkable degree of flexibility in the P domains that is not genus specific or related to differences between rVLPs and authentic virions. The simplest explanation for the role of this transition is that it gives the P domains flexibility that may be used to optimize interactions with cell receptors during attachment and entry. In this way, the P domains can increase their avidity for the cell surface by being more facile in adapting to the presentation of cellular recognition motifs.     

Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species

Background

Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group.

Results

Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes - genes present in more than one strain but not in all strains - was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence.

Conclusions

Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Abdominal wall and labial edema presenting in a girl with Henoch-Schönlein purpura: a case report

Introduction

We report a rare case of gastrointestinal perforation following dacarbazine infusion for metastatic melanoma. The condition is attributed to a responding malignant melanoma in the gastrointestinal tract.

Case presentation

A 52-year-old Caucasian man presented with abdominal pain and distension, malaise, night sweats, dysphagia and early satiety. A computed tomography scan showed massive ascites, lymphadenopathy and liver lesions suspect for metastases. An upper gastrointestinal endoscopy was performed and revealed multiple dark lesions of 5 mm to 10 mm in his stomach and duodenum. When his skin was re-examined, an irregular pigmented lesion over the left clavicle measuring 15 mm × 8 mm with partial depigmentation was found. Histological examination of a duodenal lesion was consistent with a diagnosis of metastatic melanoma. The patient deteriorated and his level of lactate dehydrogenase rapidly increased. The patient was started on systemic treatment with dacarbazine 800 mg/m² every three weeks and he was discharged one day after the first dose. On the sixth day he was readmitted with severe abdominal pain. A chest X-ray showed the presence of free intraperitoneal air that was consistent with gastrointestinal perforation. His lactate dehydrogenase level had fallen from 6969U/L to 1827U/L, supporting the conclusion that the response of gastrointestinal metastases to dacarbazine had resulted in the perforation of the patient's bowel wall. A laparotomy was discussed with the patient and his family but he decided to go home with symptomatic treatment. He died 11 days later.

Conclusion

Melanoma can originate in, as well as metastasize to, the gastrointestinal tract. Gastrointestinal perforations due to responding tumors are a well-known complication of systemic treatment of gastrointestinal lymphomas. However, as the response rate of metastatic melanoma to dacarbazine is only 10% to 20%, and responses are usually only partial, perforation due to treatment response in metastatic melanoma is rare. Medical oncologists should be aware of the risk of bowel perforation after starting cytotoxic chemotherapy on patients with gastrointestinal metastases.     

Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection

Background

Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.

Methodology/Principal Findings

However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5^−/−). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5^−/− mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5^−/− BMDMs.

Conclusions/Significance

We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.     

Antibody-independent control of gamma-herpesvirus latency via B cell induction of anti-viral T cell responses

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine gamma-herpesvirus 68 (gammaHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell-/- mice during the early phase of gammaHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell-/- mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of gamma-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection.     

Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells

Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.