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The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform noncoding functions during virus replication, although an open reading frame (ORF) is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed that its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression and is more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I program, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to the wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV posttranscriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long noncoding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle.IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. The establishment of latent infection, which is lifelong and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidating how these EBV products contribute to latency is crucial for understanding EBV's role in specific malignancies and, ultimately, for clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here, we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote the continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

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The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.

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Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency.

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The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.

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Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3-and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.