Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy, a rare demyelinating disease that occurs in the setting of prolonged immunosuppression. After initial asymptomatic infection, the virus establishes lifelong persistence in the kidney and possibly other extraneural sites. In rare instances, the virus traffics to the central nervous system, where oligodendrocytes, astrocytes, and glial precursors are susceptible to lytic infection, resulting in progressive multifocal leukoencephalopathy. The mechanisms by which the virus traffics to the central nervous system from peripheral sites remain unknown. Lactoseries tetrasaccharide c (LSTc), a pentasaccharide containing a terminal α2,6-linked sialic acid, is the major attachment receptor for polyomavirus. In addition to LSTc, type 2 serotonin receptors are required for facilitating virus entry into susceptible cells. We studied the distribution of virus receptors in kidney and brain using lectins, antibodies, and labeled virus. The distribution of LSTc, serotonin receptors, and virus binding sites overlapped in kidney and in the choroid plexus. In brain parenchyma, serotonin receptors were expressed on oligodendrocytes and astrocytes, but these cells were negative for LSTc and did not bind virus. LSTc was instead found on microglia and vascular endothelium, to which virus bound abundantly. Receptor distribution was not changed in the brains of patients with progressive multifocal leukoencephalopathy. Virus infection of oligodendrocytes and astrocytes during disease progression is LSTc independent.

The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the α2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious. IMPORTANCE Infection with human JC polyomavirus (JCPyV) is common and asymptomatic in healthy individuals, but during immunosuppression, JCPyV can spread from the kidney to the central nervous system (CNS) and cause a fatal, demyelinating disease, progressive multifocal leukoencephalopathy (PML). Individuals infected with HIV, those who have AIDS, or those receiving immunomodulatory therapies for autoimmune diseases are at serious risk for PML. Recent reports have demonstrated that viral isolates from PML patients often have distinct changes within the major capsid protein. Our structural-functional approach highlights that these mutations result in abolished engagement of the carbohydrate receptor motif LSTc that is necessary for infection. Viruses with PML-associated mutations are not infectious in glial cells, suggesting that they may play an alternative role in PML pathogenesis.

JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by β-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of β-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N = 9) versus control (N = 11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

JC polyomavirus (JCV) is an important human pathogen that causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In this study we further delineate the early events of JCV entry in human glial cells and demonstrate that a pentameric subunit of the viral capsid is able to recapitulate early events in viral trafficking. We show that JCV traffics to the endoplasmic reticulum (ER) by 6h post infection, and that VP1 pentamers arrive at the ER with similar kinetics. Further, this JCV localization to the ER is critical for infection, as treatment of cells with agents that prevent ER trafficking, ER function, or ER quality control reduce JCV infectivity. These pentamers represent a new tool to study polyomavirus entry, and will be particularly useful in studying recently identified polyomaviruses that are difficult to propagate.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

JC polyomavirus (JCPyV) is a small, non-enveloped virus that persists in the kidney in about half the adult population. In severely immune-compromised individuals JCPyV causes the neurodegenerative disease progressive multifocal leukoencephalopathy (PML) in the brain. JCPyV has been shown to infect cells by both direct and indirect mechanisms, the latter involving extracellular vesicle (EV) mediated infection. While direct mechanisms of infection are well studied indirect EV mediated mechanisms are poorly understood. Using a combination of chemical and genetic approaches we show that several overlapping intracellular pathways are responsible for the biogenesis of virus containing EV. Here we show that targeting neutral sphingomyelinase 2 (nSMase2) with the drug cambinol decreased the spread of JCPyV over several viral life cycles. Genetic depletion of nSMase2 by either shRNA or CRISPR/Cas9 reduced EV-mediated infection. Individual knockdown of seven ESCRT-related proteins including HGS, ALIX, TSG101, VPS25, VPS20, CHMP4A, and VPS4A did not significantly reduce JCPyV associated EV (JCPyV(+) EV) infectivity, whereas knockdown of the tetraspanins CD9 and CD81 or trafficking and/or secretory autophagy-related proteins RAB8A, RAB27A, and GRASP65 all significantly reduced the spread of JCPyV and decreased EV-mediated infection. These findings point to a role for exosomes and secretory autophagosomes in the biogenesis of JCPyV associated EVs with specific roles for nSMase2, CD9, CD81, RAB8A, RAB27A, and GRASP65 proteins.

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis. This study utilizes super-resolution microscopy and related techniques, which surpass traditional microscopy resolution limitations, to provide both a spatial and temporal assessment of the interactions of human JC polyomavirus (JCPyV) with 5-hydroxytrypamine 2 receptors (5-HT2Rs) subtypes during viral entry. JCPyV causes asymptomatic kidney infection in the majority of the population and can cause fatal brain disease, and progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Using Fluorescence Photoactivation Localization Microscopy (FPALM), the colocalization of JCPyV with 5-HT2 receptor subtypes (5-HT2A, 5-HT2B, and 5-HT2C) during viral attachment and viral entry was analyzed. JCPyV was found to significantly enhance the clustering of 5-HT2 receptors during entry. Cluster analysis of infected cells reveals changes in 5-HT2 receptor cluster attributes, and radial distribution function (RDF) analyses suggest a significant increase in the aggregation of JCPyV particles colocalized with 5-HT2 receptor clusters in JCPyV-infected samples. These findings provide novel insights into receptor patterning during viral entry and highlight improved technologies for the future development of therapies for JCPyV infection as well as therapies for diseases involving 5-HT2 receptors.

JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.

Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive ("lytic") and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCE Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive ("lytic") and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.

The endemic human JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy in immune-suppressed patients. The mechanisms of virus infection in vivo are not understood because the major target cells for virus in the brain do not express virus receptors and do not bind virus. We found that JCPyV associates with extracellular vesicles (EVs) and can infect target cells independently of virus receptors. Virus particles were found packaged inside extracellular vesicles and attached to the outer side of vesicles. Anti-JCPyV antisera reduced infection by purified virus but had no effect on infection by EV-associated virus. Treatment of cells with the receptor-destroying enzyme neuraminidase inhibited infection with purified virus but did not inhibit infection by EV-associated virus. Mutant pseudoviruses defective in sialic acid receptor binding could not transduce cells as purified pseudovirions but could do so when associated with EVs. This alternative mechanism of infection likely plays a critical role in the dissemination and spread of JCPyV both to and within the central nervous system.IMPORTANCE JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML), a severe and often fatal neurodegenerative disease in immunocompromised or immunomodulated patients. The mechanisms responsible for initiating infection in susceptible cells are not completely known. The major attachment receptor for the virus, lactoseries tetrasaccharide c (LSTc), is paradoxically not expressed on oligodendrocytes or astrocytes in human brain, and virus does not bind to these cells. Because these are the major cell types targeted by the virus in the brain, we hypothesized that alternative mechanisms of infection must be responsible. Here we provide evidence that JCPyV is packaged in extracellular vesicles from infected cells. Infection of target cells by vesicle-associated virus is not dependent on LSTc and is not neutralized by antisera directed against the virus. This is the first demonstration of a polyomavirus using extracellular vesicles as a means of transmission.

JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People that become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or significant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins β-arrestin, adaptor protein 2 (AP2), and dynamin. Further, a β-arrestin interacting domain, the Ala-Ser-Lys (ASK) motif, within the C-terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5-HT2R ASK motifs and the activation of β-arrestin-associated proteins during internalization has not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as critical for JCPyV internalization and infectivity. Further, utilizing biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced β-arrestin binding. Utilizing small-molecule chemical inhibitors and RNA interference, G-protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between β-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and β-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and resultant infection.IMPORTANCE As intracellular parasites, viruses require a host cell to replicate and cause disease. Therefore, virus-host interactions contribute to viral pathogenesis. JC polyomavirus (JCPyV) infects most of the population, establishing a lifelong asymptomatic infection within the kidney. Under conditions of severe immunosuppression JCPyV may spread to the central nervous system, causing the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals living with HIV or undergoing immunomodulatory therapies are at risk for developing PML. The mechanisms of how JCPyV uses specific receptors on the surface of host cells to initiate internalization and infection is a poorly understood process. We have further identified cellular proteins involved in JCPyV internalization and infection and elucidated their specific interactions that are responsible for activation of receptors. Collectively, these findings illuminate how viruses usurp cellular receptors during infection, contributing to current development efforts for therapeutic options for the treatment or prevention of PML.

Polyomaviruses are small, non-enveloped DNA tumor viruses that cause serious disease in immunosuppressed people, including progressive multifocal leukoencephalopathy (PML) in patients infected with JC polyomavirus, but the molecular events mediating polyomavirus entry are poorly understood. Through genetic knockdown approaches, we identified phosphoinositide 3'-kinase γ (PI3Kγ) and its regulatory subunit PIK3R5 as cellular proteins that facilitate infection of human SVG-A glial cells by JCPyV. PI3Kα appears less important for polyomavirus infection than PI3Kγ. CRISPR/Cas9-mediated knockout of PIK3R5 or PI3Kγ inhibited infection by authentic JCPyV and by JC pseudovirus. PI3Kγ knockout also inhibited infection by BK and Merkel Cell pseudoviruses, other pathogenic human polyomaviruses, and SV40, an important model polyomavirus. Reintroduction of the wild-type PI3Kγ gene into the PI3Kγ knock-out SVG-A cells rescued the JCPyV infection defect. Disruption of the PI3Kγ pathway did not block binding of JCPyV to cells or virus internalization, implying that PI3Kγ facilitates some intracellular step(s) of infection. These results imply that agents that inhibit PI3Kγ signaling may have a role in managing polyomavirus infections.

Pneumonia is a world health problem and a leading cause of death, particularly affecting children and the elderly (1, 2). Bacterial pneumonia following infection with influenza A virus (IAV) is associated with increased morbidity and mortality but the mechanisms behind this phenomenon are not yet well-defined (3). Host resistance and tolerance are two processes essential for host survival during infection. Resistance is the host's ability to clear a pathogen while tolerance is the host's ability to overcome the impact of the pathogen as well as the host response to infection (4-8). Some studies have shown that IAV infection suppresses the immune response, leading to overwhelming bacterial loads (9-13). Other studies have shown that some IAV/bacterial coinfections cause alterations in tolerance mechanisms such as tissue resilience (14-16). In a recent analysis of nasopharyngeal swabs from patients hospitalized during the 2013-2014 influenza season, we have found that a significant proportion of IAV-infected patients were also colonized with Klebsiella oxytoca, a gram-negative bacteria known to be an opportunistic pathogen in a variety of diseases (17). Mice that were infected with K. oxytoca following IAV infection demonstrated decreased survival and significant weight loss when compared to mice infected with either single pathogen. Using this model, we found that IAV/K. oxytoca coinfection of the lung is characterized by an exaggerated inflammatory immune response. We observed early inflammatory cytokine and chemokine production, which in turn resulted in massive infiltration of neutrophils and inflammatory monocytes. Despite this swift response, the pulmonary pathogen burden in coinfected mice was similar to singly-infected animals, albeit with a slight delay in bacterial clearance. In addition, during coinfection we observed a shift in pulmonary macrophages toward an inflammatory and away from a tissue reparative phenotype. Interestingly, there was only a small increase in tissue damage in coinfected lungs as compared to either single infection. Our results indicate that during pulmonary coinfection a combination of seemingly modest defects in both host resistance and tolerance may act synergistically to cause worsened outcomes for the host. Given the prevalence of K. oxytoca detected in human IAV patients, these dysfunctional tolerance and resistance mechanisms may play an important role in the response of patients to IAV.

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

The JC and BK human polyomaviruses (JCPyV and BKPyV, respectively) establish lifelong persistent infections in the kidney. In immunosuppressed individuals, JCPyV causes progressive multifocal leukoencephalopathy (PML), a fatal neurodegenerative disease, and BKPyV causes polyomavirus-associated nephropathy (PVN). In this study, we compared JCPyV and BKPyV infections in primary human renal proximal tubule epithelial (HRPTE) cells. JCPyV established a persistent infection, but BKPyV killed the cells in 15 days. To identify the cellular factors responsible for controlling JCPyV infection and promoting viral persistence, we profiled the transcriptomes of JCPyV- and BKPyV-infected cells at several time points postinfection. We found that infection with both viruses induced interferon production but that interferon-stimulated genes (ISGs) were only activated in the JCPyV-infected cells. Phosphorylated STAT1 and IRF9, which are responsible for inducing ISGs, translocated to the nucleus of JCPyV-infected cells but did not in BKPyV-infected cells. In BKPyV-infected cells, two critical suppressors of cytokine signaling, SOCS3 and SOCS1, were induced. Infection with BKPyV but not JCPyV caused reorganization of PML bodies that are associated with inactivating antiviral responses. Blockade of the interferon receptor and neutralization of soluble interferon alpha (IFN-α) and IFN-β partially alleviated the block to JCPyV infection, leading to enhanced infectivity. Our results show that a type I IFN response contributes to the establishment of persistent infection by JCPyV in HRPTE cells.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.