How <i>P</i>. <i>falciparum</i> increases the risk of endemic Burkitt’s lymphoma.

<p>Essentially all adults are persistently infected with EBV (A). As a consequence, newly infected B cells are continually being produced that transit the GC on their way to becoming latently infected memory B cells (the site of viral persistence) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref014" target="_blank">14</a>]. Malaria is immunosuppressive (B) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref016" target="_blank">16</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref017" target="_blank">17</a>], and Torgbor et al. have shown that this results in a highly elevated throughput of EBV-infected cells in the GC (C). Torgbor et al. also showed that <i>P</i>. <i>falciparum</i> induces deregulated expression of the DNA-mutating and -cutting enzyme AID in GC cells (D). Robbiani et al. subsequently showed in a mouse model that this deregulated expression led to DNA damage, translocations, and, ultimately, lymphoma (E). Thus, infection with <i>P</i>. <i>falciparum</i> has been shown to have two effects on the GC, where eBL originates. Together, these increase the risk that a GC cell will undergo a c-myc translocation and that this cell will also be EBV-infected and, therefore, able to tolerate the translocation, synergistically increasing the likelihood that eBL will arise.</p

The <i>var2csa</i> uORF is a repressive element.

<p>A. Constructs used in transient transfections. Constructs drive expression of either the firefly <i>luciferase</i> reporter gene, <i>Renilla luciferase</i> (in the case of control plasmid HRH) or both (in the case of V2RLH). VLH contains the un-modified promoter and upstream regulatory region from <i>var7b</i>. V2LH contains the un-modified promoter and upstream regulatory region from <i>var2csa</i>. V2LHm is identical to V2LH, except a single base pair mutation has altered the start codon of the upstream open reading (uORF) from ATG to ACG. In V2BLH, the uORF has been replaced with the <i>bsd</i> coding region while in V2RLH the uORF has been replaced by the coding region for <i>Renilla luciferase</i>. In V2ΔLH the entire region upstream of the uORF, including the transcription start site, has been deleted. In SURF the uORF has been shortened to 48 bp by the introduction of a premature stop codon, while in uORFL it has been lengthened to 450 bp by eliminating the endogenous stop codon. ICL contains the intact upstream regulatory region, including the uORF, however the intercistronic region has been duplicated. B. Levels of firefly luciferase expression from each construct shown in A. C. Levels of <i>Renilla</i> luciferase expression. V2RLH supports translation of robust levels of <i>Renilla</i> luciferase indicating that the uORF can be translated. The plasmid HRH, containing the strong <i>hrp3</i> promoter, was employed as a positive control for <i>Renilla</i> luciferase expression, while V2LH, which does not contain the <i>Renilla</i> luciferase gene, was a negative control. All assays were done simultaneously in triplicate.</p

Growth rates of parasites translating different ORFs.

<p>Constructs are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000256#ppat-1000256-g004" target="_blank">Figure 4</a>. A. Parasites that have been selected for translation of the second open reading frame encoding <i>blasticidin-S-deaminase</i> (V2B+blasticidin) grow at the same rate as parasites not under blasticidin selection (V2B−blasticidin) or those in which translation of the uORF has been disrupted (V2mB+blasticidin). Parasites in which the uORF has been replaced with the coding region of <i>Renilla luciferase</i> (V2RB+blasticidin) grow at a slower rate. B. Parasites actively translating the second ORF encoding <i>blasticidin-S-deaminase</i> continue to display resistance to the drug if grown without selection pressure very briefly. C. When parasites are grown in the absence of blasticidin in the media for 21 days, they rapidly begin to repress translation of the second ORF and revert to the drug sensitive phenotype as demonstrated by slower growth when placed back under drug pressure (V2B+blasticidin). The histogram at the right shows that reversion to drug sensitivity is not due to a switch in promoter activity. Parasites grown without blasticidin pressure (V2B−BSD) for three weeks continue to express <i>bsd</i> mRNA at levels equal to or greater than those grown continuously under blasticidin pressure (V2B+BSD). <i>bsd</i> mRNA levels are shown as copy number normalized to <i>seryl-tRNA synthetase</i>.</p

Translational repression in stably transformed parasites.

<p>Parasites were stably transfected with either pVLH or pV2BLH (A). V2BLH contains the <i>bsd</i> selectable marker and VLH contains <i>hdhfr</i> for maintaining the episomes in transfected parasites (not shown). Integration into the genome occurred spontaneously and was selected for by alternate growth with or without drug. <i>luciferase</i> mRNA levels were determined using Q-RT-PCR (B) with three different primer pairs (1–3), whose corresponding location on the <i>luciferase</i> ORF is shown in (A). Levels of protein expression were assayed by measuring luciferase activity (C).</p

Evidence for translational regulation of <i>var2csa</i> in cultured, wildtype parasites.

<p>A. The parasite population NF54 VAR2CSA was selected using anti-VAR2CSA rabbit antibodies. Flow analysis indicates that a majority of the population displays VAR2CSA on the cell surface (red) with a MFI value of 14.2 compared to the background (black) with a MFI value of 5.2 (arbitrary units). Flow analysis of NF54-239 indicates that the majority of the population displays low levels of VAR2CSA on the cell surface with a MFI value of 5.0 compared to the background value of 3.6 (arbitrary units). B. Selection for VAR2CSA expression results in recognition by antibodies predominantly from female sera whereas the NF54-239 line is poorly recognized. C. <i>var</i> transcript analysis shows that <i>var2csa</i> is the dominant transcript in both the NF54 VAR2CSA (Black) and Nf54-239 (white) parasite lines.</p

Selection for reversal of translational repression by the uORF of <i>var2csa</i>.

<p>A. Constructs used for selection of parasites that translate the second open reading frame of the <i>var2csa</i> gene. The drug selectable marker <i>blasticidin-S-deaminase</i> (BSD) was used to select parasites translating the downstream cistron (equivalent to exon I of <i>var2csa</i>). All three constructs also contain the <i>hdhfr</i> selectable marker for maintaining the episomes in transfected parasites prior to blasticidin selection (not shown). B. A drug resistant population appeared after six days suggesting that a subset of parasites is capable of translating the second ORF. Replacing the uORF with <i>Renilla luciferase</i> (V2RB) led to severely retarded growth in presence of blasticidin. Analysis of episomes recovered from these parasites indicated that they had undergone recombination (not shown).</p

A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite <i>Plasmodium falciparum</i>

<div><p>Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite <i>Plasmodium falciparum</i> to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called <i>var</i> encodes the chief antigenic and virulence determinant of <i>P</i>. <i>falciparum</i> malaria. <i>var</i> genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for <i>var</i> gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that <i>var2csa</i>, an unusual and highly conserved <i>var</i> gene, occupies a unique position within the <i>var</i> gene switching hierarchy. Induction of switching through the destabilization of <i>var</i> specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of <i>var2csa</i>. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the <i>var2csa</i> promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of <i>var2csa</i> transcripts, frequent “default” switching to this locus and detection of <i>var2csa</i> untranslated transcripts in non-pregnant individuals, these data suggest that <i>var2csa</i> could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which <i>var</i> gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in <i>Plasmodium falciparum</i>.</p></div

Induction of <i>var2csa</i> in HB3 specifically activates the locus on chromosome 12.

<p>The nucleotide sequences of the two copies of <i>var2csa</i> found within the genome of HB3 are shown. Of note are the polymorphisms (surrounded by red boxes) that easily distinguish the gene found on chromosome 1 from that found on chromosome 12. In particular, two highly polymorphic regions are labeled. The location and sequence of PCR primers (red text) that amplify across these polymorphic regions are shown. Sequencing of this PCR product after induction of <i>var2csa</i> expression by chaetocin treatment or over-expression of PfSET2 dom-neg detected only the locus on chromosome 12.</p

Primer sets used in real-time PCR assays specifically to amplify <i>var2csa</i> from HB3.

<p>The numbers in parentheses are the original primer set # from Salanti et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.ref043" target="_blank">43</a>].</p><p>Primer sets used in real-time PCR assays specifically to amplify <i>var2csa</i> from HB3.</p

Effect of over-expression of PfSET2 dom-neg on <i>var</i> gene expression.

<p>(A) Schematic diagram of the domain structure of PfSET2. The top image shows the conserved domains identified using SMART (Simple Modular Architecture Research Tool, <a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>). PHD domains are shown as blue polygons while the methyltransferase domain (labeled SET) is shown in red. The SET2 Rpb1 Interacting Region (SRIR) is shown as a white triangle. (B) <i>var</i> gene expression is shown as a pie-chart, with each slice of the pie representing the fraction of the total <i>var</i> mRNA pool transcribed from each <i>var</i> gene. The left chart shows the <i>var</i> gene expression pattern in a population over-expressing firefly luciferase. This is unchanged from the untransfected population and the annotation number of the dominant gene is shown in white text. The chart on the right shows the <i>var</i> gene expression pattern in a population after over-expression of the PfSET2 dominant negative construct for 6 weeks. <i>var2csa</i> (PF3D7_1200600, shown in green) has become the dominant transcript. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s001" target="_blank">S1 Fig</a>.</p