Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA’s acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.info:eu-repo/semantics/publishedVersio

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-A crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.info:eu-repo/semantics/publishedVersio

Promising solutions for railway operations to cope with future challenges — Tackling COVID and beyond

The COVID-19 pandemic has imposed a dramatic effect on the mobility habits of both passengers and freight in the rail sector. Since the relaxation of COVID-19 restrictions worldwide, rail transport has been revitalised gradually. However, the new normal emerges with unprecedented issues, such as changed travel behaviour, lost profits, and a lack of personnel. In this paper, we determine the arising challenges due to COVID-19 and pandemics in general and subsequently propose several solutions to tackle these challenges in rail transport. These solutions cover multidisciplinary aspects such as passenger demand management, freight demand management, service design, automation, decentralisation and advanced railway technologies. By reviewing the relevant literature on COVID-19, public transport and particularly rail transport, we synthesise and identify promising lines of research that should devote more attention to a more efficient, effective and sustainable rail transport service. This paper provides policymakers, researchers, railway infrastructure managers and undertakings with an overview and an outlook for the impacts of the pandemic crisis and similar situations. It supports decision-making with more evidence and facilitates rail transport to restore its performance and reach its societal goal.Transport and PlanningTransport and Plannin

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006890.]

Macrophages drive KSHV B cell latency

Summary: Kaposi’s sarcoma herpesvirus (KSHV) establishes lifelong infection and persists in latently infected B cells. Paradoxically, in vitro B cell infection is inefficient, and cells rapidly die, suggesting the absence of necessary factor(s). KSHV epidemiology unexpectedly mirrors that of malaria and certain helminthic infections, while other herpesviruses are ubiquitous. Elevated circulating monocytes are common in these parasitic infections. Here, we show that KSHV infection of monocytes or M-CSF-differentiated (M2) macrophages is highly efficient. Proteomic analyses demonstrate that infection induces macrophage production of B cell chemoattractants and activating factor. We find that KSHV acts with monocytes or M2 macrophages to stimulate B cell survival, proliferation, and plasmablast differentiation. Further, macrophages drive infected plasma cell differentiation and long-term viral latency. In Kenya, where KSHV is endemic, we find elevated monocyte levels in children with malaria. These findings demonstrate a role for mononuclear phagocytes in KSHV B cell latency and suggest that mononuclear phagocyte abundance may underlie KSHV’s geographic disparity

Construction of EBNA-LP knockouts and their revertants.

<p><b>A.</b> Schematic representation of the EBV genome showing the latency genes and internal repeat 1 (IR1) across which the repetitive domain of EBNA-LP is transcribed. Region from the LMP genes to EBNA2 (dotted line) crosses the termini of the linear genome and is shown in <b>B.</b> Promoters Cp and Wp are indicated by bent arrows; exons are green (right facing) or purple (left facing) arrows, with intact ORFs indicated in other colours. Names of EBNA exons are shown below the relevant arrow. The splicing of latency genes is indicated above (plus strand transcripts) or below (minus strand transcripts) the exons. Two examples of the alternatively spliced EBNA transcripts are shown, indicating variable numbers of W exon pairs in EBNA-LP, variable use of Cp and Wp promoters, and differential splicing producing either EBNA2 or other downstream EBNAs. Variations in the W1 splice acceptor from C2 or W0 (red asterisks) define whether a transcript encodes EBNA2 or EBNA-LP. <b>C-E</b> Mutations introduced into recombinant viruses. Parental B95-8 sequence is shown above the introduced mutation used to generate: <b>C.</b> EBNA-LP knockout; <b>D.</b> EBNA2 knockout; <b>E.</b> EBNA-LP Y exon knockout. <b>F.</b> Flow chart showing set of recombineering steps used to generate the recombinant EBVs constructed for this study. Note that viruses whose IR1 is mutated are made via an intermediate (WKO) in which IR1 has been entirely deleted. Coloured names indicate recombinant BACs that were used to generate the viruses used in experiments–Green names are wild-type in sequence and phenotype; Red names are mutants; LPrevⁱ is shown in purple, as it contains a point change compared to wild-type that was intended to be phenotypically neutral. Alternative lab names are included for reference.</p

EBNA-LP-null LCLs only establish with a memory B cell-like phenotype.

<p><b>A.</b> Flow cytometry plots show the CD27 (x-axis) and IgD status (y-axis) of LCLs established with different viruses (top labels) in different B cell subsets (labels left) from donor B63. Numbers in the plots show the percentage of cells in each quadrant. Naïve B cell-derived LCLs were all assayed on the same day, 39 days post infection. <b>B & C.</b> Graphs showing percentage of (<b>B</b>) CD27+ve or (<b>C</b>) IgD+ve cells from LCLs (based on data from Fig 5A and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006890#ppat.1006890.s013" target="_blank">S13A Fig</a>) produced from either naïve B cells or total B cells. Data points are shown as either EBNA-LP deficient (black square) or wild-type (orange diamond). Horizontal lines show means for each group.</p