An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

ABSTRACTMetabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast (Saccharomyces cerevisiae) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell; and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing ‘free’ MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

Extensive Genetic Diversity of Polyomaviruses in Sympatric Bat Communities: Host Switching versus Coevolution

ABSTRACT Polyomaviruses (PyVs) are small DNA viruses carried by diverse vertebrates. The evolutionary relationships of viruses and hosts remain largely unclear due to very limited surveillance in sympatric communities. In order to investigate whether PyVs can transmit among different mammalian species and to identify host-switching events in the field, we conducted a systematic study of a large collection of bats (n = 1,083) from 29 sympatric communities across China which contained multiple species with frequent contact. PyVs were detected in 21 bat communities, with 192 PyVs identified in 186 bats from 15 species within 6 families representing at least 28 newly described PyVs. Surveillance results and phylogenetic analyses surprisingly revealed three interfamily PyV host-switching events in these sympatric bat communities: two distinct PyVs were identified in two bat species in restricted geographical locations, while another PyV clustered phylogenetically with PyVs carried by bats from a different host family. Virus-host relationships of all discovered PyVs were also evaluated, and no additional host-switching events were found. PyVs were identified in different horseshoe bat species in sympatric communities without observation of host-switching events, showed high genomic identities, and clustered with each other. This suggested that even for PyVs with high genomic identities in closely related host species, the potential for host switching is low. In summary, our findings revealed that PyV host switching in sympatric bat communities can occur but is limited and that host switching of bat-borne PyVs is relatively rare on the predominantly evolutionary background of codivergence with their hosts. IMPORTANCE Since the discovery of murine polyomavirus in the 1950s, polyomaviruses (PyVs) have been considered highly host restricted in mammals. Sympatric bat communities commonly contain several different bat species in an ecological niche facilitating viral transmission, and they therefore represent a model to identify host-switching events of PyVs. In this study, we screened PyVs in a large number of bats in sympatric communities from diverse habitats across China. We provide evidence that cross-species bat-borne PyV transmission exists, though is limited, and that host-switching events appear relatively rare during the evolutionary history of these viruses. PyVs with close genomic identities were also identified in different bat species without host-switching events. Based on these findings, we propose an evolutionary scheme for bat-borne PyVs in which limited host-switching events occur on the background of codivergence and lineage duplication, generating the viral genetic diversity in bats.

The Murine Polyomavirus MicroRNA Locus Is Required To Promote Viruria during the Acute Phase of Infection

ABSTRACTPolyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs duringin vivoinfections are mostly unknown. The studies presented here used murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis revealed that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection. This is consistent with the PyV miRNAs functioning to reduce viral replication during the persistent phase of infection in a natural host. Additionally, the MuPyV miRNA locus promotes viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infectingRag2−/−mice. These findings implicate the miRNA locus as functioning in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.IMPORTANCEMicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of theirin vivofunction. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of clinical relevance. Here we show a novel activity for a viral miRNA locus in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA locus in long-term persistence, it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an adaptive immune response, our work also connects thein vivoactivity of the PyV miRNA locus to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings help to better understand how the balance between PyVs and the immune response becomes altered in pathogenic states.

Murine polyomavirus microRNAs promote viruria during the acute phase of infection

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs duringin vivoinfections are mostly unknown. Studies presented here use murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis reveals that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection, indicating that during infection of a natural host, PyV miRNAs function to reduce viral replication during the persistent phase of infection. Additionally, MuPyV miRNAs promote viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/-mice. These findings implicate miRNA activity in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.ImportanceMicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of theirin vivofunction. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of possible clinical relevance. Here we show a novel activity for a viral miRNA in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA in long-term persistence, that it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an effective adaptive immune response, our work also connects thein vivoactivity of a PyV miRNA to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings may help to better understand how the balance between PyV and the immune response becomes altered in pathogenic states.

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

ABSTRACT Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.