Crimean-Congo hemorrhagic fever virus replication imposes hyper-lipidation of MAP1LC3 in epithelial cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a virus that causes severe liver dysfunctions and hemorrhagic fever, with high mortality rate. Here, we show that CCHFV infection caused a massive lipidation of LC3 in hepatocytes. This lipidation was not dependent on ATG5, ATG7 or BECN1, and no signs for recruitment of the alternative ATG12-ATG3 pathway for lipidation was found. Both virus replication and protein synthesis were required for the lipidation of LC3. Despite an augmented transcription of SQSTM1, the amount of proteins did not show a massive and sustained increase in infected cells, indicating that degradation of SQSTM1 by macroautophagy/autophagy was still occurring. The genetic alteration of autophagy did not influence the production of CCHFV particles demonstrating that autophagy was not required for CCHFV replication. Thus, the results indicate that CCHFV multiplication imposes an overtly elevated level of LC3 mobilization that involves a possibly novel type of non-canonical lipidation. Abbreviations: BECN1: Beclin 1; CCHF: Crimean-Congo hemorrhagic fever; CCHFV: Crimean-Congo hemorrhagic fever virus; CHX: cycloheximide; ER: endoplasmic reticulum; GFP: green fluorescent protein; GP: glycoproteins; MAP1LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; n.i.: non-infected; NP: nucleoprotein; p.i.: post-infection; SQSTM1: sequestosome 1.

VP24 is a molecular determinant of Ebola virus virulence in guinea pigs.

In sharp contrast to human and nonhuman primates, guinea pigs and some other mammals resist Ebola virus (EBOV) replication and do not develop illness upon virus inoculation. However, serial passaging of EBOV in guinea pigs results in a selection of variants with high pathogenicity. In this report, using a reverse genetics approach, we demonstrate that this dramatic increase in EBOV pathogenicity is associated with amino acid substitutions in the structural protein VP24. We show that although replication of recombinant EBOV carrying wild-type VP24 is impaired in primary peritoneal guinea pig macrophages and in the liver of infected animals, the substitutions in VP24 allow EBOV to replicate in guinea pig macrophages and spread in the liver of infected animals. Furthermore, we demonstrate that both VP24/wild type and the guinea pig-adapted VP24/8mc are similar in their ability to block expression of interferon-induced host genes, suggesting that the increase in EBOV virulence for guinea pigs is not associated with VP24 interferon antagonist function. This study sheds light on the mechanism of resistance to EBOV infection and highlights the critical role of VP24 in EBOV pathogenesis.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level.

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta1 and alpha5 integrins and major histocompatibility complex I molecules. The level of GP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP, expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GP-expressing cells with GP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

Ectodomain shedding of the glycoprotein GP of Ebola virus.

In this study, release of abundant amounts of the Ebola virus (EBOV) surface glycoprotein GP in a soluble form from virus-infected cells was investigated. We demonstrate that the mechanism responsible for the release of GP is ectodomain shedding mediated by cellular sheddases. Proteolytic cleavage taking place at amino-acid position D637 removes the transmembrane anchor and liberates complexes consisting of GP1 and truncated GP2 (GP(2delta)) subunits from the cell surface. We show that tumor necrosis factor alpha-converting enzyme (TACE), a member of the ADAM family of zinc-dependent metalloproteases, is involved in EBOV GP shedding. This finding shows for the first time that virus-encoded surface glycoproteins are substrates for ADAMs. Furthermore, we provide evidence that shed GP is present in significant amounts in the blood of virus-infected animals and that it may play an important role in the pathogenesis of infection by efficiently blocking the activity of virus-neutralizing antibodies.