Kaposi's Sarcoma-Associated Herpesvirus ORF45 Mediates Transcriptional Activation of the HIV-1 Long Terminal Repeat via RSK2

Abstract

Robust activation of human immunodeficiency virus type 1 (HIV-1) gene expression occurs upon superinfection with Kaposi's sarcoma-associated herpesvirus (KSHV), a common AIDS-associated pathogen. Though the mechanisms underlying this phenotype remain unknown, several KSHV-encoded factors have been reported to stimulate HIV-1 long terminal repeat (LTR) activity. Here, we systematically evaluated the ability of KSHV tegument proteins to modulate the activation of an integrated HIV-1 LTR and revealed that the most potent individual activator is ORF45. ORF45 directs an increase in RNA polymerase II recruitment to the HIV-1 LTR, leading to enhanced transcriptional output. ORF45 is a robust activator of the p90 ribosomal S6 kinases (RSK), and we found that this activity is necessary but not sufficient to increase transcription from the LTR. Of the three widely expressed RSK isoforms, RSK2 appears to be selectively involved in LTR stimulation by both KSHV ORF45 and HIV-1 Tat. However, constitutively active RSK2 is unable to stimulate the LTR, suggesting that ORF45 may preferentially direct this kinase to a specific set of targets. Collectively, our findings reveal a novel transcriptional activation function for KSHV ORF45 and highlight the importance of RSK2 in shaping the transcriptional environment during infection. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a prominent AIDS-associated pathogen. Previous studies have shown that infection of cells containing human immunodeficiency virus type 1 (HIV-1) with KSHV leads to potent stimulation of HIV-1 gene expression by activating the HIV-1 promoter, termed the long terminal repeat (LTR).Here, we compared the abilities of various KSHV proteins to activate gene expression from the HIV-1 LTR and found that KSHV ORF45 is the most potent activator. ORF45 is known to induce cell signaling through ribosomal S6 kinase (RSK) and enhance protein translation. However, we revealed that the activation of a specific isoform of RSK by ORF45 also leads to increased mRNA synthesis from the LTR by the host RNA polymerase. Collectively, our findings provide new insight into the interviral interactions between KSHV and HIV that may ultimately impact disease.

Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

“…ERK and RSK have been shown to be involved in transcriptional regulation by phosphorylation of transcription factors, histones, and histone-modifying enzymes (32). Furthermore, a role for ORF45 in transcriptional regulation is supported by the recent report from Glaunsinger's group that ORF45 potently increases HIV long terminal repeat (LTR) transcription activity in an RSK2-dependent manner (47). Because of its role in transcriptional and translational regulation, ORF45 could have a broad impact on the expression of not only viral but also cellular genes.…”

“…Second, as mentioned above, ORF45-mediated activation of RSK is involved in the direct interaction between KSHV and HIV-1, which is well known to drastically increase both the incidence and severity of KS. ORF45 has been shown to work synergistically with the HIV-1-encoded protein Tat to potently increase LTR transcription activity in an RSK-dependent manner (47,54). Conversely, Tat has been found to increase KSHV lytic gene expression and infectivity (55)(56)(57).…”

ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

“…During KSHV lytic replication in either primary infection or reactivation, transient ERK activation induced by viral particles, 12-O-tetradecanoylphorbol-13-acetate (TPA), or another stimulus activates IE expression and initiates lytic replication (11,(35)(36)(37); however, ORF45-null KSHV and an ORF45-F66A point mutant (which are deficient in sustained ERK-p90 ribosomal S6 kinase [RSK] activation) exhibit reduced lytic gene expression and progeny virion production (31,38,39), suggesting that sustained ERK-RSK activation is essential for late viral transcription. In fact, a recent study showed that ORF45 directly increases the recruitment of RNA polymerase II and enhances transcription through RSK2 (40), indicating that ORF45-RSK2 promotes late transcription.…”

A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses

“…More importantly, KSHV ORF45 has also been shown to cause persistent activation of the extracellular regulated kinase (ERK) and p90 ribosomal S6 kinases (RSKs) (13,14). This activity is important not only for virus-host interactions (15,16), but also for virusvirus interaction between KSHV and HIV (17,18).…”