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.

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated 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 localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells.

The transcriptome of the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial induction, and a comprehensive annotation of the KSHV genome was determined. A set of unique coding sequence (UCDS) features and a process to resolve overlapping transcripts were developed to accurately quantitate transcript levels from specific promoters. Similar patterns of KSHV expression were detected in BCBL-1 cells undergoing long-term latent infections and in primary latent infections of both BEC and LEC cultures. High expression levels of poly-adenylated nuclear (PAN) RNA and spliced and unspliced transcripts encoding the K12 Kaposin B/C complex and associated microRNA region were detected, with an elevated expression of a large set of lytic genes in all latently infected cultures. Quantitation of non-overlapping regions of transcripts across the complete KSHV genome enabled for the first time accurate evaluation of the KSHV transcriptome associated with viral latency in different cell types. Hierarchical clustering applied to a gene correlation matrix identified modules of co-regulated genes with similar correlation profiles, which corresponded with biological and functional similarities of the encoded gene products. Gene modules were differentially upregulated during latency in specific cell types indicating a role for cellular factors associated with differentiated and/or proliferative states of the host cell to influence viral gene expression.

KSHV attachment and entry are dependent on αVß3 integrin localized to specific cell surface microdomains and do not correlate with the presence of heparan sulfate.

Cellular receptors for KSHV attachment and entry were characterized using tyramide signal amplification (TSA)-enhanced confocal microscopy. Integrins αVß3, αVß5 and α3ß1 were detected on essentially all the actin-based cell surface microdomains that initially bind KSHV, while the presence of CD98 and heparan sulfate (HS), the putative attachment receptor, was more variable. KSHV bound to the same cell surface microdomains with and without HS indicating that initial attachment of KSHV is not dependent on HS and that receptors other than HS can mediate attachment. A human salivary gland (HSG) epithelial line was identified, which lacks αVß3 but expresses high levels of HS, α3ß1 and other putative KSHV receptors. These cells were resistant to KSHV binding and infection. Reconstitution of cell surface αVß3 rendered HSG cells highly susceptible to KSHV infection, demonstrating a critical role for αVß3 in the binding and entry of KSHV that is not shared with other proposed receptors.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry.

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells.

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.

Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor.

Kaposi's sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin alpha(3)beta(1) during virus entry. Although the ligand specificity of alpha(3)beta(1) has been controversial, current literature indicates that alpha(3)beta(1) ligand recognition is independent of RGD. We compared alpha(3)beta(1) to the RGD-binding integrin, alpha(V)beta(3), for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that beta(3)-CHO cells overexpressing alpha(V)beta(3) specifically bound gB(RGD), whereas alpha(3)-CHO cells overexpressing alpha(3)beta(1) did not. Function-blocking antibodies to alpha(V)beta(3) inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to alpha(3)beta(1) did not. Using affinity-purified integrins and confocal microscopy, alpha(V)beta(3) bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific alpha(V)beta(3) antagonists, including cyclic and dicyclic RGD peptides and alpha(V)beta(3) function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from alpha(3)-null mice lacking alpha(3)beta(1) were fully competent for infection by KSHV, and reconstitution of alpha(3)beta(1) function by transfection with alpha(3) cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of alpha(3)beta(1) on the cell surface expression of alpha(V)beta(3) and on alpha(V)beta(3)-mediated adhesion of alpha(3)-CHO cells overexpressing alpha(3)beta(1) were detected, consistent with previous reports of transdominant inhibition of alpha(V)beta(3) function by alpha(3)beta(1). These observations may explain previous reports of an inhibition of KSHV infection by soluble alpha(3)beta(1). Our studies demonstrate that alpha(V)beta(3) is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).