Ramon Flick

Two Key Residues in EphrinB3 Are Critical for Its Use as an Alternative Receptor for Nipah Virus

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1–A5 and ephrinB1–B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (Koff = 1.94 × 10−3 s−1 versus 1.06 × 10−4 s−1 for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu–Trp residues in the solvent exposed G–H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr–Met residues in the homologous positions in ephrinB1 with Leu–Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G–H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon β (IFN-β) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-β gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-β promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-β promoter chromatin structure.

Rift Valley Fever Virus

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.

Reverse Genetics System for Uukuniemi Virus (Bunyaviridae): RNA Polymerase I-Catalyzed Expression of Chimeric Viral RNAs

ABSTRACT We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of theBunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5′- and 3′-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

Mass Spectrometric Characterization of Proteins from the SARS Virus A Preliminary Report

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.

Reverse genetics for crimean-congo hemorrhagic fever virus.

ABSTRACT The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family Bunyaviridae. The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family Bunyaviridae. This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

RNA Polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

Reverse genetics technology for Rift Valley fever virus: Current and future applications for the development of therapeutics and vaccines

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.

Rescue of Hantaan virus minigenomes.

Hantavirus infections are a major public health concern worldwide. Their widespread geographical distribution and their ability to produce serious, often fatal, human disease underline the need for a system that allows manipulation of these viruses. We describe here the first successful establishment of a reverse genetics technology for Hantaan virus, the prototype of the genus Hantavirus. The system offers a unique opportunity to study the biology of hantaviruses, the pathogenesis of the diseases, and the efficacy of antiviral and prophylactic measures against hantavirus infections.

Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen, which is the cause of a tick-borne illness occurring in many areas of Africa, Asia, and Europe. CCHF is characterized by a sudden onset of high fever, chills, and severe headache. Other symptoms can include gastrointestinal disorders, such as nausea, vomiting, and diarrhea. In severe cases, hemorrhagic manifestations can occur and often present as large areas of ecchymosis, rather than frank bleeding. Exposure to ticks, particularly those in the genus Hyalomma, or direct contact with virus-infected animals or people are considered the major risk factors. Studies on CCHFV are impeded by the biocontainment needed for their manipulation. However, the increasing worldwide medical awareness, the enormous interest of the media in hemorrhagic fever diseases, and their potential to be used as a bioweapon, have greatly spurred on research on this important virus, as evidenced by many new developments including the development of a reverse genetics system which should greatly enhance future research with this virus.