The Expression and Nuclear Retention Element of Polyadenylated Nuclear RNA Is Not Required for Productive Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors that are required for lytic replication are expressed. The most abundant viral transcript is the long noncoding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNAs have diverse functions, including the regulation of gene expression and the immune response. PAN possesses two main cis-acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV bacmid containing a deletion of the 79-nucleotide (nt) ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or the production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus, and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, open reading frame 59 (ORF59), LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlight how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggest that PAN may have other functional domains apart from the ENE. IMPORTANCE KSHV is an oncogenic herpesvirus that establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with the lytic replication of the virus. PAN RNA is the most abundant viral transcript during the reactivation of KSHV and is required for viral replication. Deletion and knockdown of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the cis elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and in vitro approaches, this is the first study to demonstrate the role of the ENE in the context of infection and that the ENE of PAN is not required for the lytic replication of KSHV.

Keywords: ENE; Kaposi’s sarcoma-associated herpesvirus; PAN RNA; lncRNA.

FIG 1

Major structural features and viral protein binding regions for PAN. (A) PAN cis-acting MRE (blue) and ENE (green) regions. (Top) Major viral protein interaction domains within PAN. (Bottom) PAN RNA secondary structure model of the 3 major stem-loops of the MRE, including the ORE, MRE core, and ENE triple helix showing a poly(A) tail. (B) Whole-genome sequence mapping reads showing the PAN sequences in WT and ΔENE viruses compared to the KSHV reference genome as presented in Qiagen CLC Genomics Workbench. The ENE-specific deletion is shown.

FIG 2

The ENE does not affect the expression of IE, E, and L viral transcripts. RNA was harvested and purified from iSLK BAC16 ORF59-HA WT, ΔENE, and ΔPAN cells at 6, 24, 48, and 72 hpi. Purified RNA was DNase treated and used for cDNA synthesis followed by qPCR using primers specific for ORF50, PAN, ORF58/59, ORF9, ORF57, ORF K2, ORF K13, and ORF26. Results represent data from three independent experiments. Bar graphs represent means ± standard deviations (SD). A one-way ANOVA (analysis of variance) nonparametric Freidman test was performed (P value of <0.05).

FIG 3

The PAN ENE facilitates nuclear retention. (A) RNA was extracted from nuclear and cytoplasmic fractions at 24 and 72 hpi. RNA was treated with DNase and used for cDNA synthesis and qPCR using primers/probes specific for 7SK, PAN, and ORF26. Bar graphs represent means ± SD. Two-way ANOVA Sidak’s multiple-comparison test was used (P value of <0.05). (B) Western blot showing protein expression in nuclear and cytoplasmic fractions.

FIG 4

PAN localizes as nuclear punctate spots in the absence of the ENE. (Top) FISH was performed using biotin oligonucleotides specific for either PAN or LacZ as a negative control in iSLK BAC16 ORF59-HA WT and ΔENE cell lines at 12, 24, and 48 hpi. PAN is shown (red), nuclei are stained with DAPI (blue), and merged images are shown. (Bottom) LacZ as a negative control.

FIG 5

ORF59, ORF57, LANA, H1.4, and H2A retain the ability to interact with PAN in the absence of the ENE. RNA-IPs were performed on iSLK BAC16 ORF59-HA ΔENE cells treated with NaB, Dox, and TPA for 48 h. Protein-RNA complexes were immunoprecipitated using antibodies specific for ORF59-HA, ORF57, LANA, H1.4, or H2A. RNA was isolated and used as the template for RT-PCR using primers specific for either PAN, U1, or GAPDH.

FIG 6

Production of infectious virus in BAC16ΔENE. (A) Expression of viral proteins in iSLK BAC16 ORF59-HA WT and ΔENE cells at 24 and 72 hpi. (B) Total DNA was harvested at 6, 24, 48, and 72 hpi. DNA was purified and used in qPCRs with KSHV-specific primers/probes to determine the relative levels of viral DNA accumulation compared to normalized cellular DNA and untreated samples. (C) The ENE does not affect infectious virus production. Quantification of infectious virus was performed as described in Materials and Methods. Statistical significance was determined by an unpaired t test (P value of <0.05). Results represent data from three independent experiments. NS, not significant. (D) Rate of replication of WT compared to ΔENE virus. Induced cells at the indicated time points were harvested, and viral genomes were quantified by qPCR using KSHV-specific primers/probes. Experiments were performed in triplicate.