Measles Virus Bearing Measles Inclusion Body Encephalitis-Derived Fusion Protein Is Pathogenic after Infection via the Respiratory Route.

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host.

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

Aggresome formation by the adenoviral protein E1B55K is not conserved among adenovirus species and is not required for efficient degradation of nuclear substrates.

Much of the work on the basic molecular biology of human adenoviruses has been carried out on a very limited number of the more than 60 serotypes, primarily the highly related species C viruses adenovirus type 5 (Ad5) and Ad2 and, to some extent, Ad12 of species A. Until recently, it has been widely assumed that insights obtained with these model viruses were representative of all human adenoviruses. Recent studies on the E3 ubiquitin ligase formed by the viral E1B55K and E4orf6 proteins with a cellular Cullin-based complex indicated that although all species form such a functional complex, significant variations exist in terms of complex composition and the substrates that are degraded. In the present report we conducted a comprehensive analysis of the localization of E1B55K products from representatives of six of the seven adenovirus species in the presence and the absence of the corresponding E4orf6 protein. We found that although in some species E1B55K localized in aggresomes, such was not always the case, suggesting that these structures are not necessary for the efficient degradation of substrates. In addition, differences were evident in the localization of E1B55K, although all forms readily associated with PML. Finally, Ad5 E1B55K was seen to localize in close proximity to Rab11, a marker for the endosomal recycling compartment, and both focused at the microtubule organizing center. These findings suggest that E1B55K from some species may employ the transport system utilized by the membrane recycling pathway to assemble aggresomes and the possibility that this structure might then affect recycling of cell surface components.

Reovirus replication protein µ2 influences cell tropism by promoting particle assembly within viral inclusions.

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein µ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of µ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.

The cellular chaperone hsc70 is specifically recruited to reovirus viral factories independently of its chaperone function.

Mammalian orthoreoviruses replicate and assemble in the cytosol of infected cells. A viral nonstructural protein, µNS, forms large inclusion-like structures called viral factories (VFs) in which assembling viral particles can be identified. Here we examined the localization of the cellular chaperone Hsc70 and found that it colocalizes with VFs in infected cells and also with viral factory-like structures (VFLs) formed by ectopically expressed µNS. Small interfering RNA (siRNA)-mediated knockdown of Hsc70 did not affect the formation or maintenance of VFLs. We further showed that dominant negative mutants of Hsc70 were also recruited to VFLs, indicating that Hsc70 recruitment to VFLs is independent of the chaperone function. In support of this finding, µNS was immunoprecipitated with wild-type Hsc70, with a dominant negative mutant of Hsc70, and with the minimal substrate-binding site of Hsc70 (amino acids 395 to 540). We identified a minimal region of µNS between amino acids 222 and 271 that was sufficient for the interaction with Hsc70. This region of µNS has not been assigned any function previously. However, neither point mutants with alterations in this region nor the complete deletion of this domain abrogated the µNS-Hsc70 interaction, indicating that a second portion of µNS also interacts with Hsc70. Taken together, these findings suggest a specific chaperone function for Hsc70 within viral factories, the sites of reovirus replication and assembly in cells.

A subpopulation of arenavirus nucleoprotein localizes to mitochondria.

Viruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

Curcumin Inhibits Replication of Human Parainfluenza Virus Type 3 by Affecting Viral Inclusion Body Formation.

Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.

Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase.

The minimal virus requirements for the generation of influenza virus-like particle (VLP) assembly and budding were reassessed. Using neuraminidase (NA) from the H5N1 and H1N1 subtypes, it was found that the expression of NA alone was sufficient to generate and release VLPs. Biochemical and functional characterization of the NA-containing VLPs demonstrated that they were morphologically similar to influenza virions. The NA oligomerization was comparable to that of the live virus, and the enzymic activity, whilst not required for the release of NA-VLPs, was preserved. Together, these findings indicate that NA plays a key role in virus budding and morphogenesis, and demonstrate that NA-VLPs represent a useful tool in influenza research.

Negri bodies and other virus membrane-less replication compartments.

Viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. Particularly, viral replication and assembly often take place in viral factories where specific viral and cellular proteins as well as nucleic acids concentrate. Viral factories can be either membrane-delimited or devoid of any cellular membranes. In the latter case, they are referred as membrane-less replication compartments. The most emblematic ones are the Negri bodies, which are inclusion bodies that constitute the hallmark of rabies virus infection. Interestingly, Negri bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. This is a paradigm shift in the field of virus replication. Here, we review the different aspects of membrane-less virus replication compartments with a focus on the Mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells.

Prevalence of inclusion body disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium.

Inclusion body disease (IBD) is caused by reptarenaviruses and constitutes one of the most notorious viral diseases in snakes. Although central nervous system disease and various other clinical signs have been attributed to IBD in boid and pythonid snakes, studies that unambiguously reveal the clinical course of natural IBD and reptarenavirus infection are scarce. In the present study, the prevalence of IBD and reptarenaviruses in captive snake collections and the correlation of IBD and reptarenavirus infection with the clinical status of the sampled snakes were investigated. In three IBD positive collections, long-term follow-up during a three- to seven-year period was performed. A total of 292 snakes (178 boas and 114 pythons) from 40 collections in Belgium were sampled. In each snake, blood and buffy coat smears were evaluated for the presence of IBD inclusion bodies (IB) and whole blood was tested for reptarenavirus RNA by RT-PCR. Of all tested snakes, 16.5% (48/292) were positive for IBD of which all were boa constrictors (34.0%; 48/141) and 17.1% (50/292) were reptarenavirus RT-PCR positive. The presence of IB could not be demonstrated in any of the tested pythons, while 5.3% (6/114) were reptarenavirus positive. In contrast to pythons, the presence of IB in peripheral blood cells in boa constrictors is strongly correlated with reptarenavirus detection by RT-PCR (P<0.0001). Although boa constrictors often show persistent subclinical infection, long-term follow-up indicated that a considerable number (22.2%; 6/27) of IBD/reptarenavirus positive boas eventually develop IBD associated comorbidities.

Mutations within A 35 amino acid region of P6 influence self-association, inclusion body formation, and Caulimovirus infectivity.

Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1-D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection.

Characterization of retroviruses from patients with multiple sclerosis.

These studies were performed to characterize retroviruses found in cell lines spontaneously developed from peripheral blood mononuclear cells (PBMNC) from 6 multiple sclerosis patients, a patient with progressive myelopathy and a healthy control. The cell lines are B-lymphoblastoid and produce Epstein-Barr virus (EBV) particles or express EBV proteins. The B-lymphoblastoid cell lines are also characterized by production of low, fluctuating amounts of retrovirus. The low productivity complicates purification and characterization, but implementation of product-enhanced reverse transcriptase (PERT) assays has provided a highly useful tool for monitoring retrovirus production. By electron microscopy, the retroviral particles appear type-C-like. Functional assays indicate the presence of Pol, Gag and Env. Indirect ELISA demonstrates a significant relation between disease activity and reactivity towards retroviral peptides. Molecular characterization is primarily based on RT-PCR, cloning, sequencing and Northern- or Southern analyses. Molecular characterization is continuing.

Cloning and sequencing of the gene for an enhancing factor from Pseudaletia separata entomopoxvirus.

An enhancing factor (EF) has been shown to increase the susceptibility of the armyworm, Pseudaletia separata, to this nuclear polyhedrosis virus. Using amino acid microsequencing data obtained from the purified EF protein, we cloned and sequenced the gene for EF. The deduced amino acid sequence exhibited a significant similarity to major spindle proteins, fusolins, whose function is unknown, from Heliothis armigera, Choristoneura biennis and Melolontha melolontha entomopoxviruses.

Nicotiana benthamiana plants transformed with the plum pox virus helicase gene are resistant to virus infection.

Nicotiana benthamiana Domin. plants were transformed with the cytoplasmic inclusion protein (CI) gene of plum pox potyvirus (PPV) to investigate, whether this non-structural protein would be able to confer resistance. The CI protein is an RNA helicase, which contains a conserved nucleotide binding motif (NTBM) and plays an important role in viral replication. Two gene constructions were developed for plant transformation. The first contains the original coding sequence of the CI gene under the control of 35S-promoter and nos terminator signal, the second is mutated in the NTBM region. Several transgenic plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. The integration of the viral genes into the plant genome was confirmed using the polymerase chain reaction and the transgene derived mRNAs were detected by Northern blot hybridization. The CI protein in the transgenic plants could not be detected by Western blot analyses. One transgenic line containing the mutated CI gene remained completely symptomless after PPV infection, indicating that the putative defective helicase gene was capable of eliciting virus resistance.

Molecular cloning, expression, and purification of nuclear inclusion A protease from tobacco vein mottling virus.

The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.

A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here, we report a rapid and simple method for the purification of copia VLPs from Drosophila Schneider 2 (S2) cells. The VLP purification procedure consists of a series of short centrifugation steps and eliminates the tedious and time-consuming performance of sucrose, metrizamide and cesium chloride (CsCl) gradients. The purity and presence of VLPs at different purification steps was monitored by transmission electron microscopy (TEM), immunochemical detection methods, and by Northern blot analysis. In addition to providing a fast protocol for VLP purification, our results also show that copia particles are mainly located in the nucleus of S2 cells. This new protocol may find broad applications for the purification of various other VLPs, and thus, may be of great value to other investigators with similar interests.

Specific detection of monkeypox virus by polymerase chain reaction.

The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72-95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences.

Restricted virus protein translation in canine distemper virus inclusion body polioencephalitis.

In this study, inclusion body polioencephalitis, an uncommon form of canine distemper virus (CDV)-induced encephalitis, was investigated for viral protein and mRNA expression by immunohistochemistry (IH) and in situ hybridization and, in addition, infiltrating cells were characterized by IH. Lesions were predominantly found in the grey matter of the brain stem and the immune response, dominated by T cells, was associated with a strong MHC II upregulation. Abundant expression of all viral protein mRNAs and reduced or lacking protein translation, especially of the matrix protein were the most important findings, indicating that restricted virus infection in the grey matter might represent a mechanism for viral persistence in distemper polioencephalitis.