The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

Replication-competent recombinant vesicular stomatitis viruses (rVSVs) expressing the type I transmembrane glycoproteins and selected soluble glycoproteins of several viral hemorrhagic fever agents (Marburg virus, Ebola virus, and Lassa virus) were generated and characterized. All recombinant viruses exhibited rhabdovirus morphology and replicated cytolytically in tissue culture. Unlike the rVSVs with an additional transcription unit expressing the soluble glycoproteins, the viruses carrying the foreign transmembrane glycoproteins in replacement of the VSV glycoprotein were slightly attenuated in growth. Biosynthesis and processing of the foreign glycoproteins were authentic, and the cell tropism was defined by the transmembrane glycoprotein. None of the rVSVs displayed pathogenic potential in animals. The rVSV expressing the Zaire Ebola virus transmembrane glycoprotein mediated protection in mice against a lethal Zaire Ebola virus challenge. Our data suggest that the recombinant VSV can be used to study the role of the viral glycoproteins in virus replication, immune response, and pathogenesis.

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV-and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein.In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA. Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae.Their genome consists of a singlestranded RNA genome of negative polarity with a length of about 19 kb. The filovirus genome is transcribed in monocistronic mRNA species, which encode seven structural proteins: a single surface protein (GP), a matrix protein (virus protein 40 [VP40]), a second minor matrix protein (VP24), and four nucleocapsid proteins (nucleoprotein [NP], L protein, VP35, and VP30) (7,15,16,18,25). Both EBOV and MARV cause severe hemorrhagic fever in humans and nonhuman primates (4,19). Infections with filoviruses are characterized by high fever, hemorrhages, and shock (19,22). For Zaire-EBOV and MARV, mortality rates up to 90% have been described; the mortality rates for Sudan-EBOV are about 60% (from the Centers for Disease Control and Prevention website [http: //www.cdc.gov/]). To date, neither a vaccine nor a therapy for treating infected patients is available.In contrast to many other viruses, no host cell proteins with antiviral activity have been identified so far for filoviruses. However, it is known that filoviruses antagonize the interferon (IFN) response (17), suggesting that the IFN pathway plays a role in the host cell response against filoviruses. Both VP35 and VP24 of EBOV have been found to be involved in the IFN antagonism (6,12,23).In an attempt to search for host cell antiviral proteins active against filoviruses, we analyzed the effect of the CCCH-type zinc finger antiviral protein (ZAP) (8) on EBOV and MARV replication. ZAP was discovered via its ability to inhibit Moloney murine leukemia virus (MMLV)...

The highly pathogenic enveloped Marburg virus (MARV) is composed of seven structural proteins and the nonsegmented negative-sense viral RNA genome. Four proteins (NP, VP35, VP30, and L) make up the helical nucleocapsid, which is surrounded by a matrix that is composed of the viral proteins VP40 and VP24. VP40 is functionally homologous to the matrix proteins of other nonsegmented negative-strand RNA viruses. As yet, the function of VP24 remains elusive. In the present study we found that VP24 colocalized with inclusions in MARV-infected cells that contain preformed nucleocapsids and with nucleocapsids outside the inclusions. Coexpression studies revealed that VP24 is recruited into the inclusions by the presence of NP. Furthermore, VP24 displayed membrane-binding properties and was recruited into filamentous virus-like particles (VLPs) that are induced by VP40. The incorporation of VP24 altered neither the morphology of VLPs nor the budding efficiency of VLPs. When VP24 was silenced in MARV-infected cells by small interfering RNA technology, the release of viral particles was significantly reduced while viral transcription and replication were unimpaired. Our data support the idea that VP24 is essential for a process that takes place after replication and transcription and before budding of virus progeny. It is presumed that VP24 is necessary for the formation of transport-competent nucleocapsids and/or the interaction between the nucleocapsids and the budding sites at the plasma membrane.

Our results indicate that DC-SIGN is not an EBOV receptor but, rather, is an attachment-promoting factor that boosts entry into B cell lines susceptible to low levels of EBOV GP-mediated infection.

The nucleocapsid protein VP35 of Marburgvirus, a filovirus, acts as the cofactor of the viral polymerase and plays an essential role in transcription and replication of the viral RNA. VP35 forms complexes with the genome encapsidating protein NP and with the RNA-dependent RNA polymerase L. In addition, a trimeric complex had been detected in which VP35 bridges L and the nucleoprotein NP. It has been presumed that the trimeric complex represents the active polymerase bound to the nucleocapsid. Here we present evidence that a predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homo-oligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (i) the probability to form coiled coils, (ii) homo-oligomerization, and (iii) the function of VP35 in viral RNA synthesis. Further, it was found that homo-oligomerization-negative mutants of VP35 could not bind to L. Thus, it is presumed that homo-oligomerization-negative mutants of VP35 are unable to recruit the polymerase to the NP/RNA template. In contrast, inability to homo-oligomerize did not abolish the recruitment of VP35 into inclusion bodies, which contain nucleocapsid-like structures formed by NP. Finally, transcriptionally inactive mutants of VP35 containing the functional homo-oligomerization domain displayed a dominant-negative phenotype. Inhibition of VP35 oligomerization might therefore represent a suitable target for antiviral intervention.Marburgvirus (MARV) and the closely related Ebolavirus (EBOV) together make up the family Filoviridae, which is classified in the order Mononegavirales. MARV causes a fulminant hemorrhagic fever in humans and nonhuman primates with high fatality rates (28). To date, neither a vaccine nor a curative treatment for MARV infection in humans is available. However, live attenuated recombinant vaccines have been described which protected nonhuman primates against MARV and EBOV infections (23). These might represent promising candidate vaccines for human use as well. The recent outbreak of MARV disease in Angola underlines the emerging potential of this pathogen (4). The MARV particle is composed of seven structural proteins. Four of them, NP, VP35, VP30, and L, constitute the nucleocapsid complex of MARV, which surrounds the viral genome. NP, the major nucleocapsid protein, self-assembles into tubular nucleocapsid-like structures, which are found intracellularly in large inclusions (25,29). The formation of the tubular structures by NP is presumed to represent the first step in the assembly of the nucleocapsid. NP interacts with VP35, which in turn interacts with the RNAdependent RNA polymerase L (3). The complex of VP35 and L represents the active RNA-dependent RNA polymerase, with VP35 serving as a polymerase cofactor (33). Additionally, a trimeric complex was observed consisting of NP, VP35, and L, with VP35 connecting L and NP (3). Three of the four nucleocapsid proteins, NP, VP35, and L, are essential for transcription and replication of the viral ...

BackgroundMarburg virus (MARV), a zoonotic pathogen causing severe hemorrhagic fever in man, has emerged in Angola resulting in the largest outbreak of Marburg hemorrhagic fever (MHF) with the highest case fatality rate to date.Methodology/Principal FindingsA mobile laboratory unit (MLU) was deployed as part of the World Health Organization outbreak response. Utilizing quantitative real-time PCR assays, this laboratory provided specific MARV diagnostics in Uige, the epicentre of the outbreak. The MLU operated over a period of 88 days and tested 620 specimens from 388 individuals. Specimens included mainly oral swabs and EDTA blood. Following establishing on site, the MLU operation allowed a diagnostic response in <4 hours from sample receiving. Most cases were found among females in the child-bearing age and in children less than five years of age. The outbreak had a high number of paediatric cases and breastfeeding may have been a factor in MARV transmission as indicated by the epidemiology and MARV positive breast milk specimens. Oral swabs were a useful alternative specimen source to whole blood/serum allowing testing of patients in circumstances of resistance to invasive procedures but limited diagnostic testing to molecular approaches. There was a high concordance in test results between the MLU and the reference laboratory in Luanda operated by the US Centers for Disease Control and Prevention.Conclusions/SignificanceThe MLU was an important outbreak response asset providing support in patient management and epidemiological surveillance. Field laboratory capacity should be expanded and made an essential part of any future outbreak investigation.