Negative Strand Viruses

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin-α7 is a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Here, we elucidate the mechanisms by which influenza A viruses may escape replicative restriction in the absence of importin-α7. To address this question, we assessed viral evolution in mice lacking the importin-α7 gene. We show that three mutations in particular occur with high frequency in the viral NP protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-α7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin-α7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin-α7 to replicative fitness in human cells.

The Paramyxoviridae and Pneumoviridae families include important human and animal pathogens. To identify common host factors, we performed genome-scale siRNA screens with wild-type-derived measles, mumps, and respiratory syncytial viruses in the same cell line. A comparative bioinformatics analysis yielded different members of the coatomer complex I, translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. A more detailed characterization of ABCE1 revealed its essential role for viral protein synthesis. Taken together, these data sets provide new insight into the interactions between paramyxoviruses and pneumoviruses and host cell proteins and constitute a starting point for the development of broadly effective antivirals.

Purpose: To report an unusual association between posterior scleritis and cat scratch disease. Design: Observational case report. Methods: Review of the clinical, laboratory, photographic, angiographic, OCT scans, ultrasound and orbital MRI records of a patient with posterior scleritis secondary to cat scratch disease. Results: A 44- year- old female patient, with no history of inflammatory or systemic diseases, developed a rapid progressive loss of vision in her left eye associated with mild pain and redness. Best corrected visual acuity was at 8/20 in the left eye and 20/20 in the right eye. The mobilization of the eye ball was remarkably painfull. There was no afferent pupillary defect. Slitlamp examination showed a conjunctival hyperemia and a mild inflammation of the anterior chamber. Fundus examination revealed optic disc edema and choroidoretinal folds at the posterior pole. The right eye was normal. B- mode ultrasound examination revealed a thickened sclera with T-sign. Fluo...

Despite the long history of more than three thousand years, the knowledge about rabies is still rather incomplete. In 1584, the Italian Girolamo Fracastoro stated in his text "The Incurable Wound" the observation that infected humans have to be regarded as inevitably moribund once clinical symptoms manifest, which unfortunately remains true until now, more than four hundred years later. In 1885, Louis Pasteur generated the first vaccine against the disease, although he did not know about the actual nature of its causative agent, for which he defined the word 'virus' (Latin for 'poison'). Building upon the work of the German scientist Georg Gottfried Finke, who transferred rabies from a rabid dog by sprinkling infectious saliva into wounds in 1804, and Pierre Victor Galtier, who described in 1879 the adaptation and the 3.1.3. Epidemiology Rabies is the most important viral zoonosis [265], although humans are usually a deadend host. The virus is endemic in a plethora of hosts throughout the world except Antarctica and some island countries, for example Greenland, New Zealand, Iceland and Japan. In most of the developed countries however, at least the contamination of domestic animals with RV is under control due to the application of consequent vaccination strategies [146]. Globally, canines present the major reservoir and vector of RV and pose a serious health hazard especially in Asia, Latin America and Africa. Here, community and stray dogs are common, and often nobody feels responsible for their vaccination. Regionally, different RV If post-exposure prophylaxis (PEP) cannot be administered immediately, a delay up to five days may be justifiable [161]. First symptoms of rabies usually manifest after an incubation period of one to two months. However, time spans between infection and outbreak of seven days up to six years have been documented [343], although the latter are extremely rare. The prodrome is rather unspecific, resembling a common cold, with fever, headache and general malaise, but this stage usually indicates the end of the ability to prevent a fatal outcome by PEP. After infection, RV progresses centripetally within nerve fibers from the site of entry into the CNS, causing often numbness, itching and neuropathic pain in the affected skin region [163]. After a few days up to one week, patients enter an acute neurological phase, which can manifest in three forms. People suffering from encephalic (furious) rabies develop the classical aggressive behavior and hyperactivity, which is the most common reception people have of rabies. Other possible symptoms include cerebral dysfunctions such as attention deficiency, sleeplessness, hallucinations, aerophobia and the already mentioned hydrophobia, as well as autonomic dysfunctions like hypersalivation, hyperventilation, abnormal pupil reactions, and generalized goose skin. Symptoms may deteriorate by thirst, anxiety, light, noise and other triggers. After only a few days, the patient lapses into coma and eventually dies due to cardiorespiratory failure. The second clinical form of the infection, paralytic (dumb) rabies, causes an ascending paralysis by involving peripheral nerves and is likely to be mixed up with Guillain-Barré syndrome (GBS), an autoimmune disorder of unknown cause that has the same effects [335]. But unlike GBS, RV infection leads inevitably to death, although the survival period is usually longer than in the furious manifestation of the disease [169]. The reason why a patient develops the furious or the dumb form of rabies is still unknown, but it is assumed that the RV strain, the location of the entry site, the incubation period and the virus localization in the CNS as well as the genetic background of the patient have an influence on it [164, 168, 270]. Often, patients, who contracted the virus from bats, present with atypical symptoms, for example neuropathic pain, myoclonus, hemiparesis or ataxia. These cases of non-classical

Sendai virus (SeV) HN protein is dispensable for virus particle production. HN incorporation into virions strictly depends on a cytoplasmic domain SYWST motif. HNAFYKD, with SYWST replaced with the analogous sequence of measles virus (MeV) H (AFYKD), is not incorporated in virus particles produced by LLCMK2 cells, although it is normally expressed at the plasma membrane. Unlike HNSYWST, HNAFYKD is not internalized to late endosomes, raising the possibility that HN internalization is required for uptake into virus particles. Various mosaic MeV-H containing increasing amounts of the SeV-HN all failed to be taken up in SeV virions. However, when co-expressed with HNAFYKD these MeV-H chimera induced HNAFYKD uptake into virions showing that internalization is not a prerequisite for HN uptake into particles. We propose that HN incorporation in virus particles requires first neutralization by HN of a putative inhibitor of infectious particle formation.

The cellular tail-interacting 47-kDa protein (TIP47) acts positively on HIV-1 and vaccinia virus production. We show here that TIP47, in contrast, acts as a restriction factor for Sendai virus production. This conclusion is supported by the occurrence of increased or decreased virus production upon its suppression or overexpression, respectively. Pulse-chase metabolic labeling of viral proteins under conditions of TIP47 suppression reveals an increased rate of viral protein synthesis followed by increased incorporation of viral proteins into virus particles. TIP47 is here described for the first time as a viral restriction factor that acts by limiting viral protein synthesis.

The Paramyxovirus membrane associated proteins are composed of two integral membrane glycoproteins, HN (H, G) and F, and of a matrix protein (M) carpeting the membrane inner layer. For Sendai virus (SeV), F and M have been proposed to form a complex at the endoplasmic reticulum that further migrates to the cell periphery where it represents a nucleation site for viral assembly completion (Essaidi-Laziosi et al., 2013). HN is recruited in the assembly complex once expressed at the cell surface. In contrast to F and M, HN appears dispensable for virus particle production. However, upon F suppression, concomitant HN suppression restricts viral particle production much more severely than F suppression alone, suggesting that HN plays a role as well. In this study, we demonstrate that the transmembrane and cytoplasmic regions of F are sufficient to promote virus particle production and incorporation of a foreign protein in viral particles. We further identify in the F cytoplasmic tail the site of interaction with M. We next confirm HN participation in viral particle production and we provide genetic evidence for a participation of M in the process. We finally derive observations that may provide a mechanism by which the viral C protein participates in viral particle production by mediating HN-M interaction.

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extracellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F 5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F 5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F 5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface.

I The abbreviations used am: IFN, interferon; IFN-4, y (type 11, immune) interferon; VSV, vesicular stomatitis virus; NaDodS04, sodium dodecyl sulfate; EGTA, ethylene glycol bis(8-aminoethyl EXPERIMENTAL PROCEDURES AND RESULTS~ DISCUSSION We have examined in detail the molecular basis of the antiviral state induced against VSV in human amnion U cells Portions of this paper (including "Experimental Procedures," "Results," Tables I and 11, and Figs. 1-6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3249, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Influenza infections cause several million cases of severe respiratory illness, hospitalizations, and hundreds of thousands of deaths globally. Secondary infections are a leading cause of influenza's high morbidity and mortality, and significantly factored into the severity of the 1918, 1968, and 2009 pandemics. Furthermore, there is an increased incidence of other respiratory infections even in vaccinated individuals during influenza season. Putative mechanisms responsible for vaccine failures against influenza as well as other respiratory infections during influenza season are investigated. Peripheral blood mononuclear cells (PBMCs) are used from influenza vaccinated individuals to assess antigen‐specific responses to influenza, measles, and varicella. The observations made in humans to a mouse model to unravel the mechanism is confirmed and extended. Infection with influenza virus suppresses an ongoing adaptive response to vaccination against influenza as well as other respir...

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined r...

The inhibition of viral reproduction caused by previous exposure of cells to another virus is popularly known as viral interference or superinfection resistance. The primary virus infecting the cell prevents the replication of secondary virus in that cell, thus making viral interference a very important phenomenon in viral world. It may be simplified by stating that the virus can suppress the shedding of a new virus of the homologous or heterologous type which enters the cell. The mechanisms proposed to explain viral interference may be briefly described as competing by attachment interference where receptors for the superinfecting virus are reduced or blocked, competing for intracellular components required for replication of the host machinery and virus induced interferon interference. The intensity of viral interference however depends on several factors such as adaptation of viruses to host species, pathogenicity of viruses, time of co-infection, and environmental factors. This review highlights the importance of testing of interfering viruses during the molecular screening and viral isolation attempts of infected poultry flocks and other animals so as to identify the consequences of interference during coinfection.

The inhibition of viral reproduction caused by previous exposure of cells to another virus is popularly known as viral interference or superinfection resistance. The primary virus infecting the cell prevents the replication of secondary virus in that cell, thus making viral interference a very important phenomenon in viral world. It may be simplified by stating that the virus can suppress the shedding of a new virus of the homologous or heterologous type which enters the cell. The mechanisms proposed to explain viral interference may be briefly described as competing by attachment interference where receptors for the superinfecting virus are reduced or blocked, competing for intracellular components required for replication of the host machinery and virus induced interferon interference. The intensity of viral interference however depends on several factors such as adaptation of viruses to host species, pathogenicity of viruses, time of co-infection, and environmental factors. This ...

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e92 15th ICID Abstracts / International Journal of Infectious Diseases 16S (2012) e2-e157 for migration of alleles from a "ghost" population that occurred between the 2-and 11-month samples within the viral population history of one twin. This migration is interpreted to be a reinfection event (a phenomenon which has previously been difficult to identify and quantify in CMV infections). We are currently examining migrant tracts from the ghost population and calculating the timing of the reinfection event. Conclusion: This study provides a highly detailed model of the population dynamics of CMV in congenital infections and is an important case study of the applicability of population genetic inference modeling to illuminate medically important parameters of viral infections.

Measles virus is a highly infectious RNA virus belonging to the genus Morbillivirus in the family Paramyxoviridae. It causes measles, a respiratory disease that is one of the leading causes of childhood mortality worldwide. The virus has a pleomorphic structure and a genome consisting of a single strand of negative-sense RNA. The genome encodes six structural proteins, including the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and the large polymerase (L) proteins. Measles virus enters host cells through the interaction of the viral H protein with cellular receptors, followed by fusion of the viral envelope with the host cell membrane. The virus then undergoes replication and assembly, leading to the release of new virus particles. The viral life cycle is complex and involves numerous host factors. Diagnosis of measles virus is typically made by detecting the virus in clinical specimens using laboratory tests such as RT-PCR or serological assays. ...

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid–inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an “antiviral state.” In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represen...

Bone marrow stromal antigen 2 (BST-2/tetherin) is a cellular membrane protein that inhibits the release of HIV-1. We show for the first time, using infectious viruses, that BST-2 also inhibits egress of arenaviruses but has no effect on filovirus replication and spread. Specifically, infectious Lassa virus (LASV) release significantly decreased or increased in human cells in which BST-2 was either stably expressed or knocked down, respectively. In contrast, replication and spread of infectious Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) were not affected by these conditions. Replication of infectious Rift Valley fever virus (RVFV) and cowpox virus (CPXV) was also not affected by BST-2 expression. Elevated cellular levels of human or murine BST-2 inhibited the release of virus-like particles (VLPs) consisting of the matrix proteins of multiple highly virulent NIAID Priority Pathogens, including arenaviruses (LASV and Machupo virus [MACV]), filoviruses (ZEBOV and MARV), and paramyxoviruses (Nipah virus). Although the glycoproteins of filoviruses counteracted the antiviral activity of BST-2 in the context of VLPs, they could not rescue arenaviral (LASV and MACV) VLP release upon BST-2 overexpression. Furthermore, we did not observe colocalization of filoviral glycoproteins with BST-2 during infection with authentic viruses. None of the arenavirus-encoded proteins rescued budding of VLPs in the presence of BST-2. Our results demonstrate that BST-2 might be a broad antiviral factor with the ability to restrict release of a wide variety of human pathogens. However, at least filoviruses, RVFV, and CPXV are immune to its inhibitory effect.

MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid-inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with incre...

Background Nipah virus (NiV) causes recurrent outbreaks of lethal respiratory and neurological disease in Southeast Asia. The World Health Organization considers the development of an effective vaccine against NiV a priority. Methods We produced two NiV vaccine candidates using the licensed VSV-EBOV vaccine as a backbone and tested its efficacy against lethal homologous and heterologous NiV challenge with Nipah virus Bangladesh and Nipah virus Malaysia, respectively, in the African green monkey model. Findings The VSV-EBOV vaccine expressing NiV glycoprotein G (VSV-NiVG) induced high neutralising antibody titers and afforded complete protection from homologous and heterologous challenge. The VSV-EBOV vaccine expressing NiV fusion protein F (VSV-NiVF) induced a lower humoral response and afforded complete homologous protection, but only partial heterologous protection. Both vaccines reduced virus shedding from the upper respiratory tract, and virus replication in the lungs and central nervous system. None of the protected animals vaccinated with VSV-NiVG or VSV-NiVF showed histological lesions in the CNS, but one VSV-NiVFvaccinated animal that was not protected developed severe meningoencephalitis. Interpretation The VSV-NiVG vaccine offers broad protection against NiV disease.

Zoonotic viruses, such as coronaviruses, the Ebola virus, the Zika virus, Nipah, Laasa, and rabies, can be transmitted from animals to humans (1). There is a need to develop inhibitors because they can potentially prevent or treat viral infections in humans and animals (2). Metadichol® is a nanoemulsion of long-chain alcohols that is a potent inhibitor of viruses. We present results of in vitro assays showing that it inhibits Laasa, rabies, and Nipah viruses at concentrations of 0.8 to 2.6 µg/ml. It is likely that the binding of metadichol to VDR (vitamin D receptor) leads to regulation of c-MYC (MYC Proto-Oncogene, BHLH Transcription Factor (which controls expression of SP1(SP1 transcription factor), which is the key step to controlling the viral replication gene GSPT1(G1 To S Phase Transition 1). Since metadichol is commercially available and nontoxic, with an LD50 of more than 5000 mg/kg in rats, it could be useful in the treatment of such zoonotic diseases.

By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine wheth...

The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC-MS/MS). The influenza A strains were identified to the subtype level within 3 h with MALDI-TOF MS and 6 h with LC-MS/MS, excluding the culturing time. The sensitivity of LC-MS/MS was higher compared to MALDI-TOF MS. In addition, LC-MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.

We have previously described the isolation of attenuated vaccinia viruses after long-term passage in persistently infected Friend ecythroleukemia cells. To study the potential use of these viruses as expression vectors of foreign antigens, the F gene of human respiratory syncytial vfrus was introduced into the genome of UR (wild type) and M7 (attenuated) vaccinia strains. Upon infectfon of permissive cells with these reconbfnants, the F protein was synthesized, processed and localized in the cell surface similarly to what is observed in RS virus infected cells. Also, protein F levels induced by the wild type and attenuated recombinants were similar. Experiments are in progress to determine the immunogenic potential of WR and M7 derived recombinants and their capacity to protect mice against RS virus challenge.

We evaluated pandemic infl uenza A (H1N1) 2009 virus isolates and respiratory tissues collected at autopsy by electron microscopy. Many morphologic characteristics were similar to those previously described for infl uenza virus. One of the distinctive features was dense tubular structures in the nuclei of infected cells.

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RN...

Respiratory syncytial virus is the major respiratory pathogen of infants and children worldwide, with no effective treatment or vaccine available. Steady progress has been made in understanding the respiratory syncytial virus life cycle and the consequences of infection, but many areas of respiratory syncytial virus biology remain poorly understood, including the role of subcellular localisation of respiratory syncytial virus gene products such as the matrix protein in the infected host cell. The matrix protein plays a central role in viral assembly and, intriguingly, has been observed to traffic into and out of the nucleus at specific times during the respiratory syncytial virus infectious cycle. Further, the matrix protein has been shown to be able to inhibit transcription, which may be a key to respiratory syncytial virus pathogenesis. This review will focus on the role of the matrix protein in respiratory syncytial virus infection and what is known of its nucleocytoplasmic trafficking, the understanding of which may lead to new therapeutic approaches to combat respiratory syncytial virus, and/or vaccine development. Respiratory syncytial virus Human respiratory syncitial virus (RSV) belongs to the genus Pneumovirinae within the Paramyxoviridae family, order Mononegavirales (Collins et al., 2001). Other members of this genus include bovine and caprine RSV strains, the pneumonia virus of mice and the avian pneumovirus. The Paramyxoviridae family also includes the subfamily Paramyxovirinae, which includes measles, mumps, Newcastle disease (NDV) and human parainfluenza viruses, and genus Respirovirus, which includes Sendai virus. Vesicular stomatitis (VSV), a member of genus Vesiculovirus within the Rhabdoviridae family, also shows many properties in common with paramyxoviruses.

Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell-cell signaling, protein folding, and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, Rabies, Measles, and Nipah viruses. We discuss glycobiological findings by genera in alphabetical or...

Human Respiratory Syncytial Virus (HRSV) is one of the most prevalent respiratory viral pathogens among infants and toddlers under the age of two and the elderly. Two of eleven proteins of HRSV, non-structural proteins, NS1 and NS2, are not present in the viral particle but are the rst to be translated during virus replication. Previous ndings indicate a signi cant impact of NS proteins on the host immune response. Both NS proteins are linked with interferon signaling interference, e ciently blocking IFN-associated gene transcription and T-cell maturation by association with key proteins on the IFN-signaling pathway. Here, we demonstrated that viral non-structural proteins genes themselves cause differential expression of a number of human host genes, including those of cytokines (TNF-α, IL-6), a component of AP-1 transcription factor (FOSB), and long non-coding RNAs (MALAT1 and RP11-510N19.5). Thus, our results imply that NS proteins' impact goes beyond IFN-I signaling pathways and further into immune response regulation, especially in the case of lncRNA. It also provides new data on HRSV and host cell interplay and exposes viable and previously undiscussed ways of viral interference with the host immune system.

Human Respiratory Syncytial Virus (HRSV) is one of the most prevalent respiratory viral pathogens among infants and toddlers under the age of two and the elderly. Two of eleven proteins of HRSV, non-structural proteins, NS1 and NS2, are not present in the viral particle but are the first to be translated during virus replication. Previous findings indicate a significant impact of NS proteins on the host immune response. Both NS proteins are linked with interferon signaling interference, efficiently blocking IFN-associated gene transcription and T-cell maturation by association with key proteins on the IFN-signaling pathway. Here, we demonstrated that viral non-structural proteins genes themselves cause differential expression of a number of human host genes, including those of cytokines (TNF-α, IL-6), a component of AP-1 transcription factor (FOSB), and long non-coding RNAs (MALAT1 and RP11-510N19.5). Thus, our results imply that NS proteins' impact goes beyond IFN-I signaling p...

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties o...

Respiratory syncytial virus (RSV) infection induces an oxidizing environment linked to increased viral load, expression of pro-inflammatory genes, and excessive lung inflammation. The mechanisms of how reactive oxygen species (ROS) promotes viral gene expression have remained largely elusive. Here we show that nascent (n)RNAs of RSV acquire 8-oxo-7,8-dihydroguanine (8-oxo(r)Gua) -a covalently modified guanine base in their 5’-UTR peritranscriptionally, while paired with the 3’-terminus of viral gene(s). 8-oxo(r)Gua is bound by 8-oxoguanine DNA glycosylase1 (OGG1), a complex that physically interacts with and recruits the anti-terminator protein M2-1 to increase viral gene transcription. Knockdown of OGG1 (but not other DNA glycosylases) or inhibition of its binding, significantly decreased RSV mRNA, protein levels and yield of progeny in cultured cells and airways. Collectively, these data suggest that Gua oxidation in vRNA, serves as an epitranscriptomic mark that repurposes OGG1 t...

Respiratory syncytial virus (RSV) infection induces an oxidizing environment linked to increased viral load, expression of pro-inflammatory genes, and excessive lung inflammation. The mechanisms of how reactive oxygen species (ROS) promotes viral gene expression have remained largely elusive. Here we show that nascent (n)RNAs of RSV acquire 8-oxo-7,8-dihydroguanine (8-oxo(r)Gua) -a covalently modified guanine base in their 5’-UTR peritranscriptionally, while paired with the 3’-terminus of viral gene(s). 8-oxo(r)Gua is bound by 8-oxoguanine DNA glycosylase1 (OGG1), a complex that physically interacts with and recruits the anti-terminator protein M2-1 to increase viral gene transcription. Knockdown of OGG1 (but not other DNA glycosylases) or inhibition of its binding, significantly decreased RSV mRNA, protein levels and yield of progeny in cultured cells and airways. Collectively, these data suggest that Gua oxidation in vRNA, serves as an epitranscriptomic mark that repurposes OGG1 t...

Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic β- and γ-actin patterns during the course of Sendai virus infections in polarized cells. We observed a spectacular remodeling of the β-cytoplasmic actin which correlated with productive viral multiplication. Conversely, suppression of M during the course of a productive infection resulted in the decrease of particle production and the absence of β-actin remodeling. As concomitant suppression of β- and γ-actins resulted as well in reduction of virus particle production, we propose that Sendai virus specifically induces actin remodeling in order to promote efficient virion production. Beta- and γ-cytoplasmic actin recruitment could substitute for that of the endosomal sorting complex required for transport (ESCRT) mobilized by other enveloped viruses but apparently not used by Sendai virus.

Background Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. Results Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared wi...

Importin-a adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-a3, one of the main NF-kB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-a3 promoter activity is regulated by TNF-a-induced NF-kB in a concentration-dependent manner. High-level TNFa-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-a3 mRNA expression in primary lung cells. Importin-a3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-a levels. Importin-a3-deficient mice show reduced NF-kBactivated antiviral gene expression and increased influenza lethality. Thus, importin-a3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-a3 availability in the lung might provide a new strategy to combat respiratory virus infections.

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP FluB suggest that functional NP FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP FluB. Further analysis of NP FluB indicated that it does not affect nuclear import of NP FluA. Taken together, these findings suggest a novel role of NP FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.

Sequence analysis of the nucleoprotein (NP) of swine-origin influenza virus H1N1 (S-OIV) reveals a number of atypical characteristics including an early start codon and a highly conserved, non-aromatic residue at position 313. Using an in vitro viral polymerase reconstitution assay, we found that the polymerase complex containing the NP of S-OIV (NP S-OIV) yielded substantially lower activity than those assayed with NP derived from other influenza virus strains. Moreover, alteration of the early start codon or introduction of an aromatic residue at position 313 (V313Y) did not increase but instead exacerbated the poor polymerase activity. Interestingly, when NP S-OIV was allowed to compete with that of a mouseadapted influenza virus (A/PR/8/34) to form progeny virions, only progeny bearing NP S-OIV were produced, despite the low polymerase activity associated with NP S-OIV. Our results indicated that NP S-OIV requires both the early start codon and the V313 residue for its optimal function. These characteristics are required for a strong compatibility between the S-OIV polymerase subunits and its indigenous NP over that of other strains, which might explain why productive reassortment between S-OIV and seasonal influenza viruses has yet to occur in nature.