Intracellular Transport and Processing of the Marburg Virus Surface Protein in Vertebrate and Insect Cells

AbstractThe surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oligosaccharides leading to a mature protein of 170–200 kDa approximately 30 min after pulse labeling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slightly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein

Intracellular Events and Cell Fate in Filovirus Infection

Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis

Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells)

Recent advances in marburgvirus research [version 1; peer review: 3 approved]

Marburgviruses are closely related to ebolaviruses and cause a devastating disease in humans. In 2012, we published a comprehensive review of the first 45 years of research on marburgviruses and the disease they cause, ranging from molecular biology to ecology. Spurred in part by the deadly Ebola virus outbreak in West Africa in 2013–2016, research on all filoviruses has intensified. Not meant as an introduction to marburgviruses, this article instead provides a synopsis of recent progress in marburgvirus research with a particular focus on molecular biology, advances in animal modeling, and the use of Egyptian fruit bats in infection experiments

Human and murine IFIT1 proteins do not restrict infection of negative-sense RNA viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae families

UNLABELLED: Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5\u27-triphosphates (5\u27-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5\u27-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5\u27 ends of IAV gene segments. The affinity for 5\u27-ppp RNA was approximately 10-fold lower than that for non-2\u27-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE: Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Isolation and genome characterization of Lloviu virus from Italian Schreibers’ bent-winged bats

13 Pág.Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers’ bent-winged bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its discovery. Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infection in vitro. Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding sequence of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this article is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection. These results further confirms the role of these bats as the host of this virus, possibly throughout their entire geographic range. This is an important result to further understand the role of bats as the natural hosts for zoonotic filoviruses.This work was supported by the National Research, Development and Innovation Office, Hungary under grants NKFIH FK131465 (G.K.) and FK137778 (T.G.), and RRF-2.3.1-21-2022-00010; and the National Institutes of Health under grant R21AI169646 (E.M.). T.G. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.N

Isolation of infectious Lloviu virus from Schreiber’s bats in Hungary

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber’s bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber’s bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber’s bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics

Processing of Genome 5′ Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5′ triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5′ triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5′ monophosphate-specific exonuclease indicates that the RIG-I-activating 5′ triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5′ end as a strategy of viruses to evade the innate immune response

ICTV Virus Taxonomy Profile: Filoviridae.

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae