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. 2016 Aug 12;90(17):7657-66.
doi: 10.1128/JVI.03102-15. Print 2016 Sep 1.

The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

Zhigang Zhang  1 , Wuguo Chen  1 , Marcia K Sanders  1 , Kevin F Brulois  1 , Dirk P Dittmer  1 , Blossom Damania  2
Affiliations

The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

Zhigang Zhang et al. J Virol. .

Abstract

The K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOP displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REV upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOP also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.

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Figures

FIG 1
FIG 1
Construction of wild-type or mutant K1 recombinant viruses. (A) Schematic diagram of WT K1 and mutant K1 genes. The gray bar represents a FLAG epitope tag, and each star represents a stop codon. (B) PFGE analysis of KpnI-digested DNA of BAC16 and its derivatives. DNAs of WT KSHV (BAC16) and its derivatives KSHV-K1FLAG, KSHV-K15×STOP, KSHV-K1REV, and KSHVΔK1 were digested with KpnI and analyzed by PFGE. The 29.7-kb fragment is the result of replacing the original WT K1 gene with FLAG-tagged K1 and the presence of an additional KpnI site in the FLAG-tagged K1 gene compared to the WT K1 gene. The 33.2-kb fragment in the KSHVΔK1 virus is due to the replacement of the K1 ORF with the Kan-Neo (RpsL-Neo) cassette.
FIG 2
FIG 2
K1 is required for Akt phosphorylation and lytic gene expression in reactivated cells. (A and B) A total of 5 × 105 WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells were seeded overnight and then treated with 1.5 μg/ml of doxycycline for 72 h (A) or for 24, 48, and 72 h (B). Cells were harvested, lysed, and subjected to Western blot analysis with an anti-FLAG antibody to detect K1, an anti-pAkt (S473) antibody to detect phosphorylated Akt, an anti-vIL-6 antibody to detect vIL-6, an anti-K8α antibody to detect K8α, and an anti-ORF45 antibody to detect ORF45. (C) A total of 2 × 105 cells were seeded overnight and then treated with 3 μg/ml of doxycycline for 72 h. Cells were fixed with 3.7% formaldehyde and subjected to an immunofluorescence assay with primary anti-ORF59 antibody (1:500; Advanced Biotechnologies) and secondary anti-mouse IgG antibody conjugated to tetramethyl rhodamine isocyanate (1:500). (D) Graph depicting the number of quantitated ORF59-positive cells per field following reactivation.
FIG 3
FIG 3
Small amounts of K1 protein are expressed in uninduced KSHV-infected iSLK cells. iSLK cells latently infected with KSHV-K1FLAG, KSHV-K1REV, and KSHVΔK1 were harvested and subjected to immunoprecipitation (IP) with an anti-FLAG antibody to immunoprecipitate FLAG-tagged K1 and then subjected to Western blot (WB) analysis with an anti-FLAG-HRP-conjugated antibody.
FIG 4
FIG 4
KSHV K1 is required for efficient reactivation from latency. (A and B) A total of 5 × 105 stable iSLK cells latently infected with WT KSHV, KSHV-K1FLAG, KSHV-K15×STOP, KSHV-K1REV, and KSHVΔK1 were plated and examined by fluorescence microscopy in the absence of any induction (A) or after being treated with 1 μg/ml of doxycycline in DMEM supplemented with 2% FBS for 72 h (B). Images were taken at a ×4 magnification. hpi, hours postinfection. (C) Graph depicting the number of GFP-positive cells per field that were quantitated following reactivation.
FIG 5
FIG 5
KSHV K1 is required for efficient viral replication. Stable iSLK cells latently infected with WT KSHV, KSHV-K1FLAG, KSHV-K15×STOP, KSHV-K1REV, and KSHVΔK1 were treated with 1 μg/ml of doxycycline for 72 h. Following reactivation, the supernatants (A and C) and cells (B) were harvested for qPCR to determine viral genome copy numbers. (A) KSHV genome copy numbers in the supernatants of reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells. (B) Quantification of intracellular viral genome copy numbers present in cells of reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells. For normalization across samples, intracellular viral genome copy numbers were normalized to the intracellular CT value of GAPDH. (C) KSHV genome copy numbers from DNase-treated and untreated supernatants of reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells.
FIG 6
FIG 6
KSHV K1 is required for efficient infectious virion production. Supernatants from reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells were used to infect naive Vero cells for 72 h. (A) GFP-positive cells were imaged by fluorescence microscopy at 72 h postinfection (hpi) at a ×20 magnification. (B) Graph depicting numbers of GFP-positive cells per field. (C) Supernatants from reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells were used to infect naive Vero cells for 72 h, and infected cells were analyzed by flow cytometry for the percentage of GFP-positive cells to quantitate infectivity. (D) Infected Vero cells from panel A were harvested, and the intracellular viral genome copies present in infected cells were quantified by real-time PCR. For normalization across samples, intracellular viral genome copy numbers were normalized to the intracellular CT value of GAPDH. FITC, fluorescein isothiocyanate; SSC-A, side-scattered light area.
FIG 7
FIG 7
KSHV K1 is required for efficient infectious virion production and infection of primary endothelial cells. Primary HUVECs were infected with supernatants from reactivated WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells for 72 h. (A) Images of GFP-positive infected primary HUVECs were taken by fluorescence microscopy at 72 h postinfection (hpi). (B) The graph depicts the number of GFP-positive infected primary HUVECs per field.
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