Protein co-purification with the viral replicase S. cerevisiae strain DED1::66HA-hphNT1 was generated by homologous recombination using strain BY4741. PCR was performed using plasmid pYM-16 (EUROSCARF) [73] as template and primers #2493 (GCAGAAAACGAAGAATCCT-CACCCTAGTTTGTCTGAAATCAATCGATGAATTCGAG-CTCG) / #2494 (GGCTGGGGTAACAGCGGTGGTTCAAA-CAACTCTTCTTGGTGGCGTACGCTGCAGGTCGAC). The PCR products were transformed to BY4741 and recombinant yeast colonies were selected in YPD plates supplemented with hygromycin. Recombinant yeast strains were transformed with plasmids pGBK-HisFlagp33, pGAD-HisFlagp92 and pYC-DI72 [24] . 66His/Flag-tagged HF-p33 and HF-p92 were expressed from ADH1 promoter and DI-72 transcript was under GAL1 promoter. After selection of transformed yeast on SC-ULH 2 plates, yeast were pre-grown overnight in selective media containing 2% glucose at 29uC. After centrifugation at 2,000 rpm for 3 min and washing the pellet with selective media containing 2% galactose, yeast were grown for 36 hours in SC-ULH 2 media containing 2% galactose at 23uC. 200 ml of pelleted yeast were used to affinity-purify HF-p33 and HF-p92 with anti-FLAG M2 agarose as described previously (see also Text S1) [14, 15] . HF-p33 and HF-p92 were detected with anti-His 6 antibody (1/5,000 dilution) and AP-conjugated anti-mouse antibody (1/5,000). DED1-66HA protein was detected with anti-HA antibody from rabbit (Bethyl; 1/10,000 dilution) and AP-conjugated anti-rabbit (1/10,000) followed by NBT-BCIP detection. Figure S1 Reduced TBSV replication in CFE prepared from Ded1-depleted yeast. CFEs were prepared from TET::DED1 yeast strain cultured in the absence of doxycycline (i.e., Ded1p is expressed) or in its presence (10 mg/ml) (i.e., Ded1p is depleted). The CFEs were programmed with recombinant p33/p92 and DI-72(+) repRNA as described in REF 21. Note that the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. The CFEs contained comparable amounts of host proteins (not shown). Figure S3 Ded1p mutants with ATPase activity promote plusstrand synthesis by the affinity-purified tombusvirus replicase. (A) Scheme of the tombusvirus replicase assay. Yeast with depleted Ded1p co-expressing p33 and p92 pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (2)repRNA, and they also contained purified recombinant Ded1p. (B) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p. The level of complementary RNA synthesis producing ''repRNA'' (marked as ''FL'', the full-length product, made via initiation from the 39-terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein. Note that this replicase preparation also synthesizes internal initiation products (''ii'') and 39-terminal extension products (''39TEX''). RNAse One digestion was used to confirm the replicase products: the de novo products are insensitive, while the 39TEX product changes migration after RNase treatment  Transmission of Infectious Diseases En Route to Habitat Hotspots BACKGROUND: The spread of infectious diseases in wildlife populations is influenced by patterns of between-host contacts. Habitat “hotspots” - places attracting a large numbers of individuals or social groups - can significantly alter contact patterns and, hence, disease propagation. Research on the importance of habitat hotspots in wildlife epidemiology has primarily focused on how inter-individual contacts occurring at the hotspot itself increase disease transmission. However, in territorial animals, epidemiologically important contacts may primarily occur as animals cross through territories of conspecifics en route to habitat hotspots. So far, the phenomenon has received little attention. Here, we investigate the importance of these contacts in the case where infectious individuals keep visiting the hotspots and in the case where these individuals are not able to travel to the hotspot any more. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a simulation epidemiological model to investigate both cases in a scenario when transmission at the hotspot does not occur. We find that (i) hotspots still exacerbate epidemics, (ii) when infectious individuals do not travel to the hotspot, the most vulnerable individuals are those residing at intermediate distances from the hotspot rather than nearby, and (iii) the epidemiological vulnerability of a population is the highest when the number of hotspots is intermediate. CONCLUSIONS AND SIGNIFICANCE: By altering animal movements in their vicinity, habitat hotspots can thus strongly increase the spread of infectious diseases, even when disease transmission does not occur at the hotspot itself. Interestingly, when animals only visit the nearest hotspot, creating additional artificial hotspots, rather than reducing their number, may be an efficient disease control measure. The spread of infectious diseases strongly depends on how habitat characteristics shape patterns of between-host interactions [1, 2] . In particular, habitat heterogeneity influences patterns of between-individual contacts and hence, disease dynamics [1, 3] . For example, ''habitat hotspots'', sites that attract individuals or social groups over long distances, can be visited by a large subset of a population. Around hotspots, between-individual contact rates often increase in frequency, which amplifies disease transmission. In humans, schools and working places are typical examples of hotspots and have been shown to accelerate the spread of measles, influenza and SARS [4, 5, 6] . Thus, limiting transmission at hotspots has become a promising strategy for mitigating epidemics (e.g., influenza [7] ) although the efficiency of such strategies also depends on the role hotspots plays relative to other sources of local transmission (e.g., influenza [6, 7] )