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Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus associated injury. Therefore, a critical aspect of ZIKV induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and mid-gestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly-elongated basal processes of the radial glial progenitor cells, and impairment of postmitotic neuronal migration were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.SignificanceZika virus infection has been associated with multiple pathologies of the central nervous system (CNS) including microcephaly, Guillain-Barré syndrome, lissencephaly, the loss of white and grey matter volume and acute myelitis. Using organotypic brain slice cultures, we determined that ZIKV replicates across different embryonic developmental stages, and viral infection can disrupt proper brain development leading to congenital CNS complications. These data illustrate that all lineages of ZIKV tested are neurotropic, and that infection may disrupt neuronal migration during brain development. The results expand our understanding of neuropathologies associated with congenital Zika virus syndrome.

Amino acid changes in the C’C”D region in poliovirus receptor domain 1 disrupt poliovirus binding. To examine further the role of the C’C”D region in poliovirus infection, we substituted this region of Pvr into the corresponding region of a murine homolog, nectin-2. The chimeric receptor, nectin-2Pvr(c'c"d), rendered transformed L cells susceptible to infection with poliovirus P1/Mahoney, but not with polioviruses P2/Lansing and P3/Leon, due to lack of binding. Twenty-four variants of P2/Lansing were selected that replicate in nectin-2Pvr(c'c"d)producing cell lines. Sequence analysis revealed 30 amino acid changes at 28 capsid residues. One change, K1103R, is found in nearly all isolates and is located at one end of the VP1 BC loop. Other alterations are located on the canyon surface, at the protomer interface, and along the perimeter of the canyon south wall. Unlike poliovirus-Pvr binding, the VP1 BC loop is required for infection of cells producing nectin-2Pvr(c'c"d).

The contributions of RIG-I and MDA5 receptors to sensing viruses of the Picornaviridae family were investigated. The picornaviruses encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3) are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFNβ synthesis is reduced in the absence of each sensor, while IFNα production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate functional redundancy within the innate immune response to picornaviruses.

Viral infections are sensed by pattern recognition receptors that trigger an innate immune response through the expression of interferons (IFNs) and other cytokines. Most RNA viruses are sensed by the RIG-I like receptors (RLR)s. The contributions of these receptors to sensing viruses of thePicornaviridaefamily were investigated. Encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3), picornaviruses of theCardiovirusandEnterovirusgenera, are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFNβ synthesis is reduced in the absence of each sensor, while IFNα production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate that innate sensing of similar viruses by RLRs depends upon the cell type.ImportanceEnteroviruses such as Coxsackieviruses are the most common human respiratory pathogens. The host’s innate immune response, in particular that modulated by the production of type I and III interferons, is thought to restrict picornavirus infection. Two cytoplasmic proteins, MDA5 and RIG-I, are critical for initiating the early innate immune response against these viruses. Mutations within MDA5 encoding gene have been associated with the development of severe enterovirus associated respiratory illnesses in healthy children. To further understand how the innate immune response dependent upon MDA5 and Rig-I is initiated during picornavirus infection, macrophages from mice lacking MDA5 or RIG-I were infected with Coxsackievirus B3 (CVB3) and a related animal virus. RIG-I is essential for type I IFN production during CVB3 infection; when MDA5 is present, viral titers are reduced by an IFN-independent pathway. These observations demonstrate that innate sensing of viruses by MDA5 and RIG-I depends upon the cell type.

Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant’s immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR’s miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro. Most importantly, in contrast to controls with a Δp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.

Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells., IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression in vivo. Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression.

Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored., IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3′ untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission. IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward., IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants.

Since 2014, numerous outbreaks of childhood infections with enterovirus D68 (EV-D68) have occurred worldwide. Most infections are associated with flu-like symptoms, but paralysis may develop in young children. It has been suggested that infection only with recent viral isolates can cause paralysis. To address the hypothesis that EV-D68 has recently acquired neurotropism, murine organotypic brain slice cultures, induced human motor neurons and astrocytes, and mice lacking the alpha/beta interferon receptor were infected with multiple virus isolates. All EV-D68 isolates, from 1962 to the present, can infect neural cells, astrocytes, and neurons. Furthermore, our results show that sialic acid binding does not play a role in EV-D68 neuropathogenesis. The study of EV-D68 infection in organotypic brain slice cultures, induced motor neurons, and astrocytes will allow for the elucidation of the mechanism by which the virus infection causes disease., Acute flaccid myelitis (AFM) is a rare but serious illness of the nervous system, specifically affecting the gray matter of the spinal cord, motor-controlling regions of the brain, and cranial nerves. Most cases of AFM are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice and adult ifnar knockout mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. All isolates replicated in organotypic mouse brain slice cultures, and three isolates replicated in primary murine astrocyte cultures. All four EV-D68 isolates examined caused paralysis and death in adult ifnar knockout mice. In contrast, no viral disease was observed after intracranial inoculation of wild-type mice. Six of the seven EV-D68 isolates, including two from 1962 and four from the 2014 outbreak, replicated in induced human neurons, and all of the isolates replicated in induced human astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as viruses replicated within neurons and astrocytes independent of binding to sialic acid. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage and can infect both neurons and astrocytes and that neurotropism is not a recently acquired characteristic as has been suggested. Furthermore, the results show that in mice the innate immune response is critical for restricting EV-D68 disease.

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics., IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design.

Movements of viruses from animals to humans underlie outbreaks of diseases, such as Ebola hemorrhagic fever, influenza, and Middle East respiratory syndrome. The severe acute respiratory syndrome (SARS) virus pandemic of 2003 was caused by a novel coronavirus (CoV) that originated in Chinese horseshoe bats (1). Results of sequence analyses have shown that viruses related to SARS-CoV continue to circulate in bats, but their potential for infecting humans is not known. Gazing at viral sequences has its limits; experiments need to be done. In PNAS, Menachery et al. (2) develop a framework for deriving viruses from these genome sequences and examining their potential to cause the next SARS pandemic (Fig. 1).

If you were a science professor, and you received two equally strong applications for the position of laboratory manager, one from a female, one from a male, which one would you pick? The answer (1.usa.gov/1WhFwwZ) may surprise you.

The innate immune system responds within minutes of infection to produce type I interferons and pro-inflammatory cytokines. Interferons induce the synthesis of cell proteins with antiviral activity, and also shape the adaptive immune response by priming T cells. Despite the discovery of interferons over 50 years ago, only recently have we begun to understand how cells sense the presence of a virus infection. Two families of pattern recognition receptors have been shown to distinguish unique molecules present in pathogens, such as bacterial and fungal cell wall components, viral RNA and DNA, and lipoproteins. The first family includes the membrane-bound toll-like receptors (TLRs). Studies of the signaling pathways that lead from pattern recognition to cytokine induction have revealed extensive and overlapping cascades that involve protein-protein interactions and phosphorylation, and culminate in activation of transcription proteins that control the transcription of genes encoding interferons and other cytokines. A second family of pattern recognition receptors has recently been identified, which comprises the cytoplasmic sensors of viral nucleic acids, including MDA-5, RIG-I, and LGP2. In this review we summarize the discovery of these cytoplasmic sensors, how they recognize nucleic acids, the signaling pathways leading to cytokine synthesis, and viral countermeasures that have evolved to antagonize the functions of these proteins. We also consider the function of these cytoplasmic sensors in apoptosis, development and differentiation, and diabetes.

The innate immune system senses RNA virus infections through membrane-bound Toll-like receptors or the cytoplasmic proteins RIG-I and MDA-5. RIG-I is believed to recognize the 5′-triphosphate present on many viral RNAs, and hence is important for sensing infections by paramyxoviruses, influenza viruses, rhabdoviruses, and flaviviruses. MDA-5 recognizes dsRNA, and senses infection with picornaviruses, whose RNA 5′-ends are linked to a viral protein, VPg, not a 5′-triphosphate. We previously showed that MDA-5 is degraded in cells infected with different picornaviruses, and suggested that such cleavage might be a mechanism to antagonize production of type I IFN in response to viral infection. Here we examined the state of RIG-I during picornavirus infection. RIG-I is degraded in cells infected with poliovirus, rhinoviruses, echovirus, and encephalomyocarditis virus. In contrast to MDA-5, cleavage of RIG-I is not accomplished by cellular caspases or the proteasome. Rather, the viral proteinase 3Cpro cleaves RIG-I, both in vitro and in cells. Cleavage of RIG-I during picornavirus infection may constitute another mechanism for attenuating the innate response to viral infection.

Virology encompasses a broad spectrum of topics touching upon many aspects of our everyday lives. However, appreciation of this impact is too often restricted to those who have specialized training and participate in virology research. The Phage Hunters Integrating Research and Education (PHIRE) program and the This Week in Virology (TWiV) podcast seek to bring virology to new audiences through two different approaches—direct involvement of undergraduates in discovering and genomically characterizing bacteriophages (PHIRE) and clear, accessible, and free discussions among experts of all topics in virology (TWiV). Here we discuss these two high-impact programs, the audiences that they serve, their broader impacts, and their future potential.

Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.

When viruses enter the host, they are first detected by the innate immune system, which comprises cytokines, sentinel cells (dendritic cells [DCs] and macrophages), complement, and natural killer (NK) cells. A second family of pattern recognition receptors comprises the cytoplasmic sensors of viral nucleic acids, MDA-5 (melanoma differentiation associated gene-5), RIG-I (retinoic acid-inducible gene I), and LGP2. Interferon (IFN) production after infection of cultured cells with paramyxoviruses, VSV, influenza virus, and the flavivirus Japanese encephalitis virus is impaired in fibroblasts from rig-I -1- mice. RIG- I- like receptors (RLRs) play central roles in viral recognition and induction of antiviral innate responses in cDCs, macrophages, and fibroblasts. Infection of mda-5 -1- mice revealed that this protein is critical for detection of infection with picornaviruses, but not flaviviruses, influenza viruses, or paramyxoviruses. Mice lacking the nitric oxide synthase gene have reduced macrophage activation and β-cell apoptosis and consequently reduced virus-induced diabetes. Understanding viral countermeasures not only improves one's understanding of innate sensing pathways but may also suggest avenues for therapeutic intervention. Infection of cells with several picornaviruses leads to cleavage or degradation of MDA-5 and RIG-I. We survive only because we possess powerful intrinsic, innate, and adaptive immune defense systems. The study of how picornaviruses interact with the innate immune system is in its infancy. Understanding innate signaling pathways and how viruses counteract them should be a productive area of research for many years.

Poliovirus is an ideal model for understanding how non-enveloped viruses enter cells and initiate infection. The study of entry of a wide variety of viruses reveals common themes that are the consequence of a central problem faced by all viruses in the passage from cell to cell or from host to host. The final stage of assembly for many viruses involves proteolytic processing of a virion protein. The replication cycle is initiated when poliovirus encounters the poliovirus receptor (Pvr), a transmembrane glycoprotein with three extracellular immunoglobulin (Ig)- like domains. Although there is still considerable controversy concerning the role of the two particles, the A particle may be an intermediate in the cell entry pathway, and the 80S empty particle may be the final protein product that accumulates after the RNA is released into the cytoplasm to initiate translation and replication. An attempt has been made to obtain structural “snapshots” of stable intermediates in the poliovirus cell entry pathway and to couple the structural information with the results of genetic, biophysical, and biochemical observations to fill in the gaps in the pathway. On the basis of structural, genetic, and biochemical evidence available to date, a working model for the cell entry of poliovirus, related enteroviruses, and major group rhinoviruses is proposed. An alternative model is proposed in which the transition from the initial binding complex to the tight-binding complex is characterized by movements of VP1, VP2, and VP3 that mimic the umbrella-like movements of the virion to A particle transition.

As knowledge of the early events in infection with enteroviruses is limited, this chapter focuses on studies with poliovirus but examines the emerging data on several other enteroviruses. The early events in virus infection, from virus binding to uncoating of the viral genome, are well characterized for a number of enveloped viruses. The poliovirus-receptor interaction is a particularly good model for studying virus entry because of the experimental manipulations that are possible given the known structure of the virus and our ability to mutagenize both the virus and its cellular receptor. Despite our increasing genetic and structural understanding of early events in poliovirus infection, many problems, such as the location of the uncoating event, remain unsolved. Perhaps imminent studies on the entry of echoviruses (ECVs) and coxsackieviruses, stimulated by the identification of their receptors, will provide clues. The roles of receptors in host range and pathogenesis have been extensively studied for poliovirus, but many questions, such as the basis of tissue tropism, remain. A study of the cell functions of virus receptors may provide information on their role in virus replication. It has been suggested that virus binding to cell receptors may lead to activation of cell events that lead to disease, and there is evidence that receptors may regulate virus-induced cytopathic effects. Studies of the interactions of cell receptors with their natural cell ligands may therefore provide clues about cell processes that are activated upon virus binding and govern the outcome of virus infections.

Major- and minor-group rhinoviruses enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although ep...

To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Major- and minor-group human rhinoviruses (HRV) enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group) and HRV1A (minor-group), upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF-2, CREB, CEBP-alpha) were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus.

Mouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (alpha2beta1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (alpha2beta1 3T3 cells) or the alpha2 subunit of human VLA-2 (alpha2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected alpha2 3T3 cells produce infectious virus. All EV-1-infected alpha2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected alpha2 3T3 or alpha2beta1 L cells. Since alpha2beta1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing.

In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.

The identification of a monoclonal antibody, AF3, which recognizes a single isoform of the cell surface protein CD44 and preferentially blocks binding of serotype 2 poliovirus to HeLa cells, suggested that CD44 might be an accessory molecule to Pvr, the cell receptor for poliovirus, and that it could play a role in the function of the poliovirus receptor site. We show here that only AF3 blocks binding of serotype 2 poliovirus to HeLa cells and, in contrast to a previously published report, that the anti-CD44 monoclonal antibodies A3D8 and IM7 are unable to block binding of poliovirus. To determine whether CD44 is involved in poliovirus infection, we analyzed the replication of all three serotypes of poliovirus in human neuroblastoma cells which lack or express CD44 and in mouse neuroblastoma cells which lack Pgp-1, the mouse homolog of human CD44, and which express Pvr. All three poliovirus serotypes replicate with normal kinetics and to normal levels in the absence or presence of CD44 or in the absence of Pgp-1. Furthermore, the binding affinity constants of all three poliovirus serotypes for Pvr are unaffected by the presence or absence of CD44 in the human neuroblastoma cell line. We conclude that CD44 and Pgp-1 are not required for poliovirus replication and are unlikely to be involved in poliovirus pathogenesis.

The cell receptor for poliovirus may be more than a simple ‘snare’ that attaches virus to cells. Recent results indicate that receptor binding may cause conformational changes in the virus that lead to uncoating of the viral RNA.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other immunoglobulin-like V-type domains. Analysis of stable cell lines expressing the mutant proteins revealed that virus binding is influenced by mutations in the predicted C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge do not develop cytopathic effect during poliovirus infection, suggesting that poliovirus-induced cytopathic effect may be induced by the virus-receptor interaction.

Soluble extracts of Spodoptera frugiperda cells expressing the poliovirus receptor (PVR) induce the native poliovirus (PV) to "A" particle conformational change (J. Virol. 64, 4697-4702). We describe the variables that regulate this passage and study the suitability of solubilized PVR both for use as an in vitro system to characterize the receptor-mediated conformational alteration and for the production of large amounts of altered virus for structural analysis. PVR seems to function in a stoichiometric fashion and the A particles produced appear as intact, stain excluding, spherical structures by electron microscopy, regardless of the extensive proteolysis of the capsid protein VP1, which takes place during the conversion. The products obtained, time course, and temperature and ionic strength dependence of the alteration of PV by the solubilized PVR are indistinguishable from those of the alteration that leads to productive infection in cultured cells. Therefore, solubilized PVR may provide a convenient in vitro system for further characterization of the cell entry process.

Interaction between the 40S ribosomal subunit and the IRES of hepatitis C virus (HCV) is thought to be independent of initiation proteins, while joining of the 60S ribosomal subunit, and initiation of translation is dependent upon components of the translation machinery. An established in vivo functional assay for internal initiation mediated by the HCV IRES was used to identify proteins needed for IRES dependent translation in Saccharomyces cerevisiae strains possessing alterations of the translation machinery. Internal initiation dependent upon the HCV IRES was abrogated in strains lacking eIF5B, and reduced in strains with altered eIF3, either lacking the Hcr1p subunit, a component of eIF3 not previously known to interact with HCV RNA, or possessing an amino acid change in the Rpg1p subunit. The HCV RNA-induced conformational change in the 40S subunit might affect positioning of eIF3 and lead to different interactions between the ribosome, eIF3, and the multifactor complex. HCV IRES dependent initiation was unaffected in strains in which the concentration of the initiating tRNA was reduced. Alteration of the δ subunit of eIF2B, which leads to inefficient recycling, or substitution of aspartic acid for serine 51 of eIF2α had no effect on internal initiation. Production of human Pkr inhibited HCV IRES dependent initiation in yeast. The synthesis of Pkr in yeast is known to result in high levels of eIF2α phosphorylation, increased Gcn4p synthesis, and reduced ribosomal protein production. These alterations may explain the effect of Pkr synthesis on HCV IRES dependent initiation in yeast.

Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.

A complete cDNA copy of the echovirus 6 genome was constructed. Complementary DNA was reversed transcribed from viral RNA. Subgenomic cDNAs were obtained by direct cloning and polymerase chain reactions. Full length cDNA was constructed into the Bluescript II vector (pBSII) using unique, overlapping, restriction sites of four clones. The cDNA was infectious and produced echovirus 6 particles that behaved in the same manner as the parental virus.

Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37°C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor.

Rhinoviruses are prevalent human pathogens that are associated with life-threatening acute asthma exacerbations. The innate immune response to rhinovirus infection, which may play an important role in virus-induced asthma induction, has not been comprehensively investigated. We examined the innate immune response in cells infected with human rhinovirus 1a (HRV1a). Beta interferon (IFN-β) mRNA was induced in HRV1a-infected cells at levels significantly lower than in cells infected with Sendai virus. To understand the basis for this observation, we determined whether components of the pathway leading to IFN-β induction were altered during infection. Dimerization of the transcription factor IRF-3, which is required for synthesis of IFN-β mRNA, is not observed in cells infected with HRV1a. Beginning at 7 h postinfection, IPS-1, a protein that is essential for cytosolic sensing of viral RNA, is degraded in HRV1a-infected cells. Induction of apoptosis by puromycin led to the cleavage of IPS-1, but treatment of HRV1a-infected cells with the pan-caspase inhibitor, zVAD, did not block cleavage of IPS-1. IPS-1 is cleaved in vitro by caspase-3 and by the picornaviral proteinases 2A pro and 3C pro . Expression of HRV1a and polioviral 2A pro and 3C pro led to degradation of IPS-1 in cells. These results suggest that IPS-1 is cleaved during HRV1a infection by three different proteases. Cleavage of IPS-1 may be a mechanism for evasion of the type I IFN response, leading to a more robust infection.