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Comparative Study
. 2015 Aug;89(15):7567-83.
doi: 10.1128/JVI.00924-15. Epub 2015 May 13.

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

Philipp A Ilinykh  1 , Ndongala M Lubaki  1 , Steven G Widen  2 , Lynnsey A Renn  3 , Terence C Theisen  3 , Ronald L Rabin  3 , Thomas G Wood  2 , Alexander Bukreyev  4
Affiliations
Comparative Study

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

Philipp A Ilinykh et al. J Virol. 2015 Aug.

Abstract

Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC.

Importance: This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.

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Figures

FIG 1
FIG 1
Schematic representation of the study. (Top) Genomes of viruses used for infection; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted eGFP gene is indicated by a white rectangle. (Bottom) Time points at which RNA samples were collected for deep sequencing and for quantitation of mRNA of type I and type III IFNs. Open circles indicate deep sequencing time points, and plus symbols indicate qRT-PCR time points.
FIG 2
FIG 2
(A) Formation of homotypic DC clusters following infection with the mutated EBOV (MOI = 2 PFU/cell) on day 4 postinfection (low magnification). (B) Bright-field (top) and UV (bottom) microscopy of DC infected with wt EBOV or mutated EBOV viruses (MOI = 2 PFU/cell) on day 4 postinfection (high magnification). (C) Analysis of eGFP expression in DC infected with the indicated viruses (MOI = 2 PFU/cell) by flow cytometry on days 2 and 4. (D) Growth kinetics of the indicated viruses in DC. Suspensions of DC in triplicate were infected and washed, and aliquots of the viruses were quantitated in Vero-E6 cell monolayers by plaque titration. Titers of mutated viruses that were significantly different (P < 0.05) from wt EBOV titers are indicated by asterisks. Titers of EBOV/VP35m/VP24m were significantly higher (P < 0.05) than those of EBOV/VP35m at all time points except day 5.
FIG 3
FIG 3
Quantitative analysis of viral transcripts, including the eGFP transcript, in DC (A and B) or monocytes (C) from two donors (left and right panels) infected with the indicated viruses (MOI = 2 PFU/cell) at 8 h (A) and 24 h (B and C) postinfection. The numbers of viral mRNA reads were normalized to the number of all reads in the sample library and normalized to the length of the transcripts to show the relative abundance of each transcript. For GP, the length of the full-length edited mRNA encoding GP1/2 was used for normalization. One arbitrary unit is equal to 100 reads after the normalizations.
FIG 4
FIG 4
VP35 and VP24 IRADs suppress different sets of genes. (A) Venn diagrams of the mRNAs upregulated in DC from donor 1 in response to infections with wt EBOV or the mutated EBOV. The fold upregulation is indicated at the left. (B) Venn diagrams of the mRNAs belonging to the indicated functional groups, which were upregulated above 4-fold in DC from donor 1 in response to infections with wt EBOV or the mutated EBOV.
FIG 5
FIG 5
Heat map of the mRNAs encoding markers of DC maturation.
FIG 6
FIG 6
Heat map of the mRNAs encoding cytokines.
FIG 7
FIG 7
Heat map of the mRNAs encoding chemokines (A) and chemokine receptors (B).
FIG 8
FIG 8
Heat map of the mRNAs encoding IFN and IFN receptors.
FIG 9
FIG 9
Radial plots of qRT-PCR analysis for type I and III IFN subtype expression in response to the various viruses at time points up to 120 h. Shown in log10 scale as copy number per μg of RNA. IFN-α subtypes are ordered according to a phylogenetic plot of amino acid sequence similarity.
FIG 10
FIG 10
Analysis of type I IFNs in eGFP+ and eGFPlow/− DC. (A) Flow cytometry analysis of eGFP expression in DC infected with the indicated viruses at an MOI of 2 or 10 PFU/cell. (Left) Primary flow cytometry data with DC from a representative donor at 48 h postinfection. The percentages of eGFP+ cells are indicated for each histogram. (Right) Percentages of eGFP+ DC after infection at an MOI of 10 PFU/ml were normalized to that after infection at an MOI of 2 PFU/cell (100%, dotted line) at 24 and 48 h postinfection. DC from individual donors are indicated by symbols, and the mean values are indicated by horizontal lines. No statistical difference between eGFP levels for infection at an MOI of 2 or 10 was found. (B) Levels of eGFP in the eGFPlow/− DC populations. (Left) eGFP expression by DC from a representative donor at 24 h after infection by wt EBOV or EBOV/VP35m at an MOI of 2 PFU/cell (black) or 10 PFU/cell (red). (Right) MFI values at 24 and 48 h after infections with the indicated viruses at an MOI of 2 or 10 PFU/cell or after mock infections; statistically significant differences in results compared to mock infections (P < 0.05) are indicated by black asterisks, and infections at an MOI of 10 PFU/cell, which resulted in IFN levels significantly increased (P < 0.05) over that for infections at an MOI of 2 PFU/cell, are indicated by red asterisks. (C to E) Expression of IFN-α by eGFP+ and eGFPlow/− DC. (C) Gating strategy used to analyze the levels of IFN-α in eGFP+ and eGFPlow/− DC. SSC-A, side scatter A; FSC-A, forward scatter A; FSCH, forward scatter height; PE, phycoerythrin. (D) Analysis of IFN-α2b in eGFP+ and eGFPlow/− populations of DC from a representative donor infected with the panel of viruses or mock infected. The percentages of type I IFN-positive cells are indicated for eGFP+ and eGFPlow/− populations. (E) Numbers of DC positive for IFN-α2b or total IFN-α after infection with the indicated mutated viruses are indicated as fold increases over wt EBOV levels. Values for DC from five donors are indicated by symbols, and mean values for each treatment are indicated by horizontal lines. Infections with the mutated viruses, which resulted in IFN levels that were significantly increased (P < 0.05) over that for wt EBOV, are indicated by asterisks.
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References

    1. Kuhn JH. 2008. Filoviruses: a compendium of 40 years of epidemiological, clinical, and laboratory studies, 1st ed Springer, New York, NY. - PubMed
    1. Kuhn JH, Bao Y, Bavari S, Becker S, Bradfute S, Brister JR, Bukreyev AA, Chandran K, Davey RA, Dolnik O, Dye JM, Enterlein S, Hensley LE, Honko AN, Jahrling PB, Johnson KM, Kobinger G, Leroy EM, Lever MS, Muhlberger E, Netesov SV, Olinger GG, Palacios G, Patterson JL, Paweska JT, Pitt L, Radoshitzky SR, Saphire EO, Smither SJ, Swanepoel R, Towner JS, van der Groen G, Volchkov VE, Wahl-Jensen V, Warren TK, Weidmann M, Nichol ST. 2013. Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae. Arch Virol 158:301–311. doi:10.1007/s00705-012-1454-0. - DOI - PMC - PubMed
    1. Albarino CG, Shoemaker T, Khristova ML, Wamala JF, Muyembe JJ, Balinandi S, Tumusiime A, Campbell S, Cannon D, Gibbons A, Bergeron E, Bird B, Dodd K, Spiropoulou C, Erickson BR, Guerrero L, Knust B, Nichol ST, Rollin PE, Stroher U. 2013. Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012. Virology 442:97–100. doi:10.1016/j.virol.2013.04.014. - DOI - PMC - PubMed
    1. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, Soropogui B, Sow MS, Keita S, De Clerck H, Tiffany A, Dominguez G, Loua M, Traore A, Kolie M, Malano ER, Heleze E, Bocquin A, Mely S, Raoul H, Caro V, Cadar D, Gabriel M, Pahlmann M, Tappe D, Schmidt-Chanasit J, Impouma B, Diallo AK, Formenty P, Van Herp M, Gunther S. 2014. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 371:1418–1425. doi:10.1056/NEJMoa1404505. - DOI - PubMed
    1. CDC. 2015, posting date Outbreak of Ebola in Guinea, Liberia, and Sierra Leone. http://www.cdc.gov/vhf/ebola/outbreaks/guinea/.

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