The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains

Abstract

dEbola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells

“…We found that the EBOV VP35 and VP24 cooperatively suppress maturation of infected human DC and that the mutations R312A in VP35 and, to a lesser degree, K142A in VP24 reverse this inhibition of maturation (15).…”

“…To make the recombinant EBOV carrying both the mutation R312A in VP35 and the mutation K142A in VP24 and expressing the enhanced green fluorescent protein (eGFP) gene, we used the previously constructed plasmid pEBOV-eGFP/VP35-R312A carrying the mutation in VP35 and pEBOVeGFP/VP24-K142A with the mutation in VP24 and each expressing the eGFP gene inserted between the NP and VP35 genes (15). The SalI-SacI fragment of the full-length pEBOV-eGFP/VP35-R312A plasmid, including part of the GP gene, the VP30 gene, and part of the VP24 gene, was replaced with its mutagenized copy from pEBOV-eGFP/VP24-K142A plasmid.…”

“…The SalI-SacI fragment of the full-length pEBOV-eGFP/VP35-R312A plasmid, including part of the GP gene, the VP30 gene, and part of the VP24 gene, was replaced with its mutagenized copy from pEBOV-eGFP/VP24-K142A plasmid. The EBOV/VP35m/VP24m virus was recovered as previously described (15) and propagated in Vero-E6 monolayers. Work with the EBOV full-length clone was performed in a laboratory approved by the NIH Recombinant DNA Advisory Committee.…”

“…On the other hand, dendritic cells (DC), which are the most effective antigen-presenting cells and are infected by EBOV, do not undergo normal maturation despite the infection (13)(14)(15). Because DC play a key role in initiation of the adaptive immune response by processing viral antigens and presenting them to naive lymphocytes, the deficient T cell response may be linked to the deficient and/or aberrant stimulation of T cells by the infected DC.…”

Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

“…Despite lack of disparity assessment of VP24 and VP35, 2 ebola virus' suppression of the maturation of dendritic cells of immune system with several viral proteins in co-operation (e.g., GP, VP24 and VP35) results in escaping the detection and response of host immune system. 27 Recently, for VP35, Leung et al 28 have concluded that the major determinant of their functional di®erences is not likely the conformational variations observed in the VP35 C-terminal basic domains between Reston and Zaire viruses, due to limited tolerance for amino acid variations in this domain. As for VP30, homodimers of VP30 have been observed for both Reston and Zaire viruses, and the residue sequences of both dimerization domains of VP30 are highly conserved, suggesting resistance of this dimerization domain to functionally signi¯cant variations.…”