RNASEK is required for internalization of diverse acid-dependent viruses

Viruses must gain entry into cells to establish infection. In general, viruses enter either at the plasma membrane or from intracellular endosomal compartments. Viruses that use endosomal pathways are dependent on the cellular factors that control this process; however, these genes have proven to be essential for endogenous cargo uptake, and thus are of limited value for therapeutic intervention. The identification of genes that are selectively required for viral uptake would make appealing drug targets, as their inhibition would block an early step in the life cycle of diverse viruses. At this time, we lack pan-antiviral therapeutics, in part because of our lack of knowledge of such cellular factors. RNAi screening has begun to reveal previously unknown genes that play roles in viral infection. We identified dRNASEK in two genome-wide RNAi screens performed in Drosophila cells against West Nile and Rift Valley Fever viruses. Here we found that ribonuclease kappa (RNASEK) is essential for the infection of human cells by divergent and unrelated positive- and negative-strand-enveloped viruses from the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae families that all enter cells from endosomal compartments. In contrast, RNASEK was dispensable for viruses, including parainfluenza virus 5 and Coxsackie B virus, that enter at the plasma membrane. RNASEK is dispensable for attachment but is required for uptake of these acid-dependent viruses. Furthermore, this requirement appears specific, as general endocytic uptake of transferrin is unaffected in RNASEK-depleted cells. Therefore, RNASEK is a potential host cell Achilles’ heel for viral infection.Viral pathogens are quite diverse in their replication strategies; however, all viruses must enter cells to initiate their replication cycles. The first step involves binding of virus particles to the cell surface. Such interactions can involve attachment factors, which have low affinity but concentrate viruses on the surface of cells, and receptors that intricately interact with viral envelope glycoproteins, which, in addition to binding, promote other aspects of infection such as internalization. Although a plethora of receptors and pathways can be used, most viruses take advantage of the cellular endocytic machinery and penetrate from within the cytosol (reviewed in refs. 1–3). Clathrin-mediated endocytosis, macropinocytosis, and caveolin-mediated endocytosis are the best-studied forms of uptake used by viruses. Clathrin-mediated endocytosis is the most common mechanism used by small viruses, as clathrin-coated vesicles have a diameter of 60–200 nm and can be enlarged to fit even larger particles (4, 5). This pathway is constitutive on most cells, and some viruses use preexisting clathrin-coated pits for entry (e.g., dengue virus), whereas others induce the formation of these structures (e.g., influenza virus) (6, 7). Macropinocytosis is an actin-dependent endocytic process for the nonselective uptake of nutrients in response to receptor engagement. It is the predominant pathway for many larger viruses, including vaccinia virus, but is also used by others, including influenza virus under some conditions (8–10). It also remains unclear which pathways are used by some viruses, including Rift Valley Fever virus (RVFV) (11–13).The molecular mechanisms involved in these uptake mechanisms are complex and rely on key molecules and organelles that are essential for cellular viability, as these uptake mechanisms bring nutrients and other metabolites into the cytosol for cellular growth and survival. Indeed, these processes and proteins involved are highly conserved from yeast to humans (14, 15). Depending on the virus entry requirements, some viruses fuse within early endosomal vesicles, whereas others traffic to more acidic compartments or macropinosomes for entry. Because many viruses are dependent on these endosomal trafficking pathways for entry, much effort has been made in identifying the specific cellular genes required for viral entry (16). Therapeutics targeting entry are appealing because it is the first step in the infection cycle, and many viruses use common pathways; thus, inhibition may be broadly antiviral, rather than active against only a specific virus. Furthermore, many viruses have high mutation rates and rapidly evolve resistance to therapeutics targeting virally encoded genes. Conversely, therapeutics against host encoded targets would likely be more difficult for the virus to evade.Recent advances in functional genomic technologies have facilitated the use of unbiased genome-wide RNAi screens to identify cellular genes required for viral infection (17). Such approaches allow for the discovery of otherwise unknown genes that play essential roles in infection. We recently performed such screens in insect cells against two disparate insect-borne human pathogens: the flavivirus West Nile virus (WNV) and the bunyavirus RVFV (18, 19). These are both arthropod-borne human pathogens for which there are no vaccines or therapeutics. Furthermore, these viruses are quite divergent: WNV is a flavivirus that is a globally important cause of encephalitis (20), and RVFV is a bunyavirus that causes significant morbidity and mortality in livestock and humans in Africa (21). In our screens, there were only three genes that promoted infection by both viruses: dRAB5, dSTX7, and dRNASEK (CG40127). The functions of RAB5 and STX7 have been described, but little is known about ribonuclease kappa (RNASEK). Both RAB5 and STX7 are involved in endosomal transport and have roles in viral entry (22, 23). Both WNV and RVFV are enveloped RNA viruses that require an acidic compartment for entry (12, 13, 24). RNASEK is a single-copy, 137-aa protein conserved from insects to humans (25–27) with an unknown function. We set out to determine the role of RNASEK in viral infection and found that RNASEK is required for internalization of a diverse panel of viruses of medical concern.     

Brief Report: Defensive symbiosis against giant viruses in amoebae

Protists are important regulators of microbial communities and key components in food webs with impact on nutrient cycling and ecosystem functioning. In turn, their activity is shaped by diverse intracellular parasites, including bacterial symbionts and viruses. Yet, bacteria–virus interactions within protists are poorly understood. Here, we studied the role of bacterial symbionts of free-living amoebae in the establishment of infections with nucleocytoplasmic large DNA viruses (Nucleocytoviricota). To investigate these interactions in a system that would also be relevant in nature, we first isolated and characterized a giant virus (Viennavirus, family Marseilleviridae) and a sympatric potential Acanthamoeba host infected with bacterial symbionts. Subsequently, coinfection experiments were carried out, using the fresh environmental isolates as well as additional amoeba laboratory strains. Employing fluorescence in situ hybridization and qPCR, we show that the bacterial symbiont, identified as Parachlamydia acanthamoebae, represses the replication of the sympatric Viennavirus in both recent environmental isolates as well as Acanthamoeba laboratory strains. In the presence of the symbiont, virions are still taken up, but viral factory maturation is inhibited, leading to survival of the amoeba host. The symbiont also suppressed the replication of the more complex Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean (Mimiviridae). Our work provides an example of an intracellular bacterial symbiont protecting a protist host against virus infections. The impact of virus–symbiont interactions on microbial population dynamics and eventually ecosystem processes requires further attention.     

Evidence for emergence of diverse polioviruses from C-cluster coxsackie A viruses and implications for global poliovirus eradication

The poliovirus (PV) eradication campaign is conducted on the premise that this virus, because of the lack of a zoonotic reservoir, will not reemerge once eradicated. This report examines the origin of PV using theoretical and experimental approaches. Our rooted phylogenetic analysis suggests a speciation of PV from a C-cluster coxsackie A virus (C-CAV) ancestor through mutation of the capsid that caused a receptor switch from intercellular adhesion molecule-1 to CD155, leading to a change of pathogenicity. This hypothesis is supported experimentally with chimeras generated from three different pairs of PV and C-CAV. Those carrying the PV capsid and the replication proteins of C-CAVs replicated well, whereas their reciprocal counterparts were either debilitated or dead. This phenomenon of asymmetry is observed also in recombinants between PV1 and C-CAV20, selected in tissue culture cells using a previously undescribed protocol. The recombinants are generated at frequencies of 10(-6) typical for PV interserotype recombination. Strikingly, they resemble genetically and phenotypically, including neurovirulence in CD155 transgenic mice, the large majority of circulating vaccine-derived PVs that have caused poliomyelitis outbreaks in different parts of the world. These data provide experimental evidence for C-CAVs being partners to PVs in generating diverse PV progeny by homologous recombination. They support speciation of a novel human pathogen (PV) from a pool of different human pathogens (C-CAVs). In a PV-free world without PV neutralizing antibodies, contemporary C-CAV, like their ancestor(s), could be fertile ground for a PV-like agent to emerge by mutation.     

Glycosphingolipids as receptors for non-enveloped viruses

Glycosphingolipids are ubiquitous molecules composed of a lipid and a carbohydrate moiety. Their main functions are as antigen/toxin receptors, in cell adhesion/recognition processes, or initiation/modulation of signal transduction pathways. Microbes take advantage of the different carbohydrate structures displayed on a specific cell surface for attachment during infection. For some viruses, such as the polyomaviruses, binding to gangliosides determines the internalization pathway into cells. For others, the interaction between microbe and carbohydrate can be a critical determinant for host susceptibility. In this review, we summarize the role of glycosphingolipids as receptors for members of the non-enveloped calici-, rota-, polyoma- and parvovirus families.     

Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans—OAS1, OAS2, and OAS3—synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.Critically important to understanding antiviral innate immunity is determining which host proteins are responsible for inhibiting different types of viruses. However, there are significant gaps in our knowledge about the specificity of many host antiviral proteins. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system (reviewed in ref. 1) is a case in point. OASs are pattern-recognition receptors for viral dsRNA, a common pathogen-associated molecular pattern for many types of RNA and DNA viruses. In humans, there are four OAS genes, all stimulated by IFN, but only three of these encode catalytically active proteins. OAS1, OAS2, and OAS3 contain one, two, and three core OAS units, respectively, but all three enzymes synthesize 2-5A from ATP upon binding dsRNA (2). OASL, containing one basic unit plus two ubiquitin-like domains, does not synthesize 2-5A but instead activates RIG-I signaling in response to dsRNA (3). In addition, OASs are structurally homologous to cGAS, a sensor of cytoplasmic DNA, often of microbial origin, that produces 2′,5′-cGMP-AMP activators of STING leading to type I IFN production (4).The only well-established function of 2-5A is to activate RNase L, causing endonucleolytic cleavage of viral and cellular ssRNAs, thereby blocking viral replication. Many viruses encode antagonists of the OAS–RNase L pathway, providing evidence that RNase L is a potent antiviral protein (1, 5, 6). However, far less clear is which of the catalytically active OAS species are responsible for suppressing different types of viral infections in human cells. The main obstacle has been the absence of OAS-KO models, other than for murine Oasl1 (7) and Oasl2 (8). Also, although some genetics studies conclude that polymorphisms in OAS1 are associated with susceptibility to West Nile virus (WNV) (9), prostate cancer (10), diabetes (11), multiple sclerosis (12), and other pathological conditions, there is little, if any, evidence that this susceptibility is mediated through RNase L.To study the impact of different OAS species on different viruses, we used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) gene-editing technology, which allows the convenient and efficient disruption of genes in mammalian cells (13, 14). Our results provide the surprising conclusion that, among the catalytically active forms of OAS proteins, OAS3 is mainly responsible for producing 2-5A activators of RNase L during infections by a wide range of different types of human viruses.     

Appendix abscess: a surgical giant presenting as a geriatric giant.

CASE REPORT: A women aged 102 years presented with falls and was found to have an atypical presentation of appendicitis. CONCLUSION: This illustrates the non-specific presentation of disease in old age and the importance of a careful medical assessment of people who have fallen.     

Severe anemia as the presenting manifestation of giant cell arteritis

Giant cell (temporal, cranial) arteritis (GCA) is usually confirmed in patients presenting with classic features. Those who present with atypical features often undergo prolonged evaluations until a diagnosis is established. Severe anemia as an initial manifestation of GCA has rarely been described. We describe herein 2 patients with biopsy-proven GCA who presented with severe anemia and significant weight loss, which corrected after corticosteroid therapy.     

Metastatic angiosarcoma of the liver preoperatively presenting as giant hemangioma

BACKGROUND: Hepatic angiosarcomas are rare tumors most often associated with exposure to vinyl chloride or other carcinogens. Only a few cases have been published without such a history. CASE REPORT: We report the case of a 73-year-old woman who was admitted to our medical department with unclear upper abdominal pain, thrombocytopenia and anemia. Both computed tomography and magnet resonance imaging revealed a giant hemangioma in the right liver with multiple small hemangiomas. To cure the problem of thrombocytopenia due to sequestration of blood cells in the hemangioma, we decided to resect the large tumor. Intraoperatively, however, the diagnosis of angiosarcoma with multiple metastases was made. The patient died 6 weeks after surgery. CONCLUSION: Problems in diagnosing angiosarcoma include the brief duration of antecedent symptoms, difficulties in radiological diagnosis, and patients without a history of professional exposure to carcinogens.     

Envelope glycoproteins from biologically diverse isolates of immunodeficiency viruses have widely different affinities for CD4.

The envelope glycoprotein gp120 of primate immunodeficiency viruses initiates viral attachment to CD4+ cells by binding to the CD4 antigen on host cell surfaces. However, among different CD4+ cell types, different viruses display distinct host cell ranges and cytopathicities. Determinants for both of these biological properties have been mapped to the env gene. We have quantitatively compared the CD4 binding affinities of gp120 proteins from viruses exhibiting different host cell tropisms and cytopathicities. The viral proteins were produced by using a Drosophila cell expression system and were purified to greater than 90% homogeneity. Drosophila-produced gp120 from T-cell tropic human immunodeficiency virus type 1 (HIV-1) BH10 exhibits binding to soluble recombinant CD4 (sCD4) and syncytia inhibition potency identical to that of pure authentic viral gp120. Relative to the affinity of HIV-1 BH10 gp120 for sCD4, that of dual tropic HIV-1 Ba-L is 6-fold lower, that of restricted T-cell tropic simian immunodeficiency virus mac is 70-fold lower, and that of noncytopathic HIV-2 ST is greater than 280-fold lower. Thus, viruses that utilize CD4 for infection do so by using a remarkably wide range of envelope affinities. These differences in affinity may play a role in determining cell tropism and cytopathicity.