Modified Anoikis Assay That Functionally Segregates Epstein-Barr Virus LMP1 Strains into Two Groups.

Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein 1 (LMP1). During epithelial metastasis, detached cells must overcome anoikis-induced cell death and gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension, but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line HK1 following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PXQXT motif on LMP1, which activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1, and Med+ promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and nonrecovering) and suggests that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors.IMPORTANCE LMP1 is one of the most divergent sequences in the EBV genome and phylogenetically segregates into seven LMP1 strains. The LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional-mapping studies on LMP1 support the notion that viral proteins could serve as molecular tool kits and potentially reveal sequence-associated risk factors in NPC metastatic progression.

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells.

UNLABELLED: Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 µM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells. IMPORTANCE: NPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

Control of phage Bxb1 excision by a novel recombination directionality factor.

Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47. Bxb1 gp47 is an unusual RDF in that it is relatively large (approximately 28 kDa), unrelated to all other RDFs, and presumably performs dual functions since it is well conserved in mycobacteriophages that utilize unrelated integration systems. Furthermore, unlike other RDFs, Bxb1 gp47 does not bind DNA and functions solely through direct interaction with integrase-DNA complexes. The nature and consequences of this interaction depend on the specific DNA substrate to which integrase is bound, generating electrophoretically stable tertiary complexes with either attB or attP that are unable to undergo integrative recombination, and weakly bound, electrophoretically unstable complexes with either attL or attR that gain full potential for excisive recombination.

Latent Membrane Protein 1 Is a Novel Determinant of Epstein-Barr Virus Genome Persistence and Reactivation.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that persistently infects humans, with nearly 95% seropositivity in adults. Infection in differentiating epithelia is permissive, but EBV-associated nasopharyngeal carcinoma (NPC) tumors harbor a clonal and nonproductive latent infection. However, in explanted NPC cultures and epithelial cell lines, episomal EBV genomes are frequently lost. The resulting unstable infection has hampered efforts to study the determinants of EBV persistence and latency in epithelial oncogenesis. The EBV nuclear antigen 1 (EBNA1) protein is required for tethering EBV episomes to cellular DNA and for mitotic segregation to daughter cells. Expression of EBNA1 does not ensure faithful partitioning of EBV episomes or replicons, suggesting that additional regulatory mechanisms have yet to be elucidated. The EBV latent membrane protein 1 (LMP1) is an oncogenic signaling protein expressed in latent and lytic cycles. This study identified that LMP1 contributes to the loss of EBV genomes in latently infected cells and promotes differentiation-induced lytic replication in a polarized air-liquid interface (ALI) culture model. Deletion of LMP1 in recombinantly infected 293 cells promoted the retention of EBV genomes in passaged cells, which was in part localized to a conserved PXQXT motif in the C-terminal signaling domain (CTAR1). Additionally, knockdown of LMP1 in the recombinantly infected NPC cell line HK1 resulted in decreased induction of lytic proteins and infectious EBV titers. These findings are consistent with the hypothesis that in epithelial infections, regulation of LMP1 mechanisms may be a determinant of infection outcome and a potential risk factor for EBV persistence in preneoplastic cells. IMPORTANCE Latent membrane protein 1 (LMP1) is a constitutively active oncogenic signaling protein encoded by Epstein-Barr virus (EBV). Despite monoclonal infection in cases of nasopharyngeal carcinoma (NPC), it has been difficult to reconcile the heterogeneous LMP1 protein levels detected in tumor cells. The LMP1 protein is a pleiotropic signaling protein with oncogenic potential. Findings from this study are consistent with the hypothesis that LMP1 has a role distinct from that of oncogenesis that facilitates the viral life cycle by promoting an unstable but productive infection in differentiating epithelia.

The effect of Epstein-Barr virus Latent Membrane Protein 2 expression on the kinetics of early B cell infection.

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein.

Cellular factors associated with latency and spontaneous Epstein-Barr virus reactivation in B-lymphoblastoid cell lines.

EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV.