Highly selective escape from KSHV-mediated host mRNA shutoff and its implications for viral pathogenesis

Abstract

During Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) lytic infection, many virus-encoded signaling molecules (e.g., viral G protein-coupled receptor [vGPCR]) are produced that can induce host gene expression in transiently transfected cells, and roles for such induced host genes have been posited in KS pathogenesis. However, we have recently found that host gene expression is strongly inhibited by 10-12 h after lytic reactivation of KSHV, raising the question of whether and to what extent de novo host gene expression induced by viral signaling molecules can proceed during the lytic cycle. Here, we show by microarray analysis that expression of most vGPCR target genes is drastically curtailed by this host shutoff. However, rare cellular genes can escape the host shutoff and are potently up-regulated during lytic KSHV growth. Prominent among these is human interleukin-6, whose striking induction may contribute to the overexpression of this cytokine in several disease states linked to KSHV infection.

Figure 1.

Expression of Ad-vGPCR and KSHV in TIME cells. (A) TIME cells were either mock infected or infected with Ad-vGPCR; 24 h after infection, total RNA was Northern blotted with a ³²P-labeled vGPCR DNA probe. (B) The same infected cells were examined by DAPI staining (left) or IFA with antibodies to vGPCR (right). (C) TIME cells stably expressing dsGFP were either mock infected, latently infected with KSHV, or infected with KSHV and lytically reactivated with Ad-RTA for 7 or 18 h. Each row represents the same field of cells showing either DAPI-stained nuclei (top), ORF59⁺ cells (middle), or the merged image of ORF59 and dsGFP (bottom).

Figure 2.

(A) vGPCR expression is detectable by 6 h after lytic reactivation. DNA arrays were performed on samples from TIME cells infected with Ad-RTA alone (RTA), or KSHV plus Ad-RTA for 6, 12, or 20 h. The colored bar represents an image taken from a TreeView cluster analysis, where increasing red intensity represents increasing vGPCR mRNA expression. Each array spot represented by the colored bar is shown on the right along with the median intensity of that spot. (B–D) Correlation of microarray and Northern blot data for three selected host mRNAs. DNA arrays were performed on samples from TIME cells infected with Ad-RTA alone (RTA), Ad-vGPCR alone (vGPCR), or KSHV and Ad-RTA for 6, 12, or 20 h. (left) As in A, increasing red intensity represents increasing mRNA expression of either GRO1 (B), VEGF-C (C), or DUSP-5 (D). (right) Northern blots of 100 ng of the corresponding poly(A) RNA from each indicated infection, as well as 100 ng of poly(A) RNA from TIME cells latently infected with KSHV, hybridized with ³²P-labeled GRO1 (B), VEGF-C (C), or DUSP-5 (D) DNA.

Figure 3.

HIF-1 and IL-6 are potently up-regulated during lytic KSHV infection of cells. (A) TIME cells were infected with either Ad-RTA for 48 h or KSHV plus Ad-RTA for the indicated times and harvested, and equivalent amounts of each lysate were immunoblotted with HIF-1 antibodies. (B) TIME cells were either mock infected or infected with the indicated viruses for 20 h, and total RNA was probed with either ³²P-labeled human IL-6 or hsp70 DNA. (left) Cells were heat shocked at 42.5°C for 2 h before harvest to demonstrate efficient shutoff of the hsp70 message. (C) TIME, HFF, and 293 cells were either mock infected, infected with Ad-RTA or Ad-vGPCR alone, or infected with KSHV and lytically reactivated with Ad-RTA or TPA for 24 h. The cells were incubated in serum-free media for the last 12 h of the infection, and IL-6 levels in the supernatants were measured using the BD Opti EIA human IL-6 ELISA kit. (D) 293T cells were transfected with either empty vector or a plasmid expressing GFP, GM-CSF, or IL-6 or cotransfected with plasmids expressing SOX and either GFP, GM-CSF, or IL-6 at a 4:1 ratio. Total RNA was Northern blotted using the indicated ³²P-labeled DNA probes.