Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

Abstract

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcomaassociated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localiza-

Reduction of Kaposi’s Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene

“…All the major LANA isoforms were observed in the samples; importantly, there are less LANA proteins expressed in the active CC9-LANA than in the mock-and inactive CC9-LANA-transduced cells (Fig. 6D) (47). These results demonstrated that Ad-CC9-LANA can indeed reduce KSHV latency by decreasing the number of KSHV episome in latently infected cells overtime.…”

Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

“…Confocal imaging suggests that the lytic replication compartments are two-dimensional rings and not cross sections of globular spheres. KSHV replication compartments have been described in other studies [ 57 – 60 ]. Replication compartments similar to what we observed have been described and shown to colocalize with DNA damage repair factors associate with NHEJ, including DNA-PK, ATM, and MRN components [ 57 ].…”

“…We observed rare punctate colocalization of KSHV lytic replication protein K8 with LANA rings, suggesting that these structures may be distinct from classical replication compartments described for other viruses. LANA has also been reported to accumulated in cytoplasmic foci during lytic replication cycle [ 60 ], but our experimental system using RFP-LANA may only observe full length LANA in the nucleus. While LANA is well known for its role in KSHV latent cycle replication, our new findings suggest that LANA also contributes to the structural organization of KSHV lytic cycle DNA replication.…”

Lack of CD8+ T-cell co-localization with Kaposi’s sarcoma-associated herpesvirus infected cells in Kaposi’s sarcoma tumors

“…To investigate the relationships between KSHV infected cells and TIIC, we first defined 'infected cells' as those evincing a punctate nuclear KSHV LANA staining pattern by IHC [20]. LANA is responsible for maintaining the KSHV episome in infected cells during latency and is also expressed in lytic reactivation [21].…”