Long Terminal Repeat

To identify the chromosomal coordinates of HML-2 proviruses in human DNA, we searched the most recent genome build (GRCh37/hg19, February 2009) using the UCSC BLAT program [49 (link)] for sequences related to the full-length nucleotide sequence of the K113 provirus (AY037928) [16 (link),49 (link)]. The DNA flanking individual 'hits' was manually searched for sequence with high similarity to prototypical HML-2 sequences as determined by the RepeatMasker program in the UCSC genome browser [67 ]. For each identified locus, complete nucleotide sequences were generated by extracting and concatenating the internal and LTR proviral segments. Additional BLAT searches with individual K113 genes (gag, pro, pol, and env) were performed to further identify HML-2 elements within the available genome. Complete sequence reconstruction was performed as above, with the minimum criterion for a provirus being the presence of an LTR and a "hit" matching > 50% of the length of a full gene, or two proximal genes with > 50% hits and no LTR. All full-length sequences were initially aligned to K113 using ClustalW [88 (link)], and manually edited in BioEdit v.7.0.9.0 [89 ]. The full-length sequences for the HML-2 proviruses located at 10p12.1 (K103) and 19p12 (K113) were from NCBI (accession numbers AF164611 and AY037928, respectively). We identified the K105 sequence by taking flanking sequence of the K105 solo LTR and searching the chimpanzee database. We identified a BAC with a provirus starting at position 74813 (AC195095.2). We found a sequence with 99% similarity through a BLAST search of the NCBI database that corresponded to a human provirus labeled K111 (GU476554). Due to the high similarity between Chimpanzee K105 and this human "K111" as well as similarity between K105 deposited 5' and 3' LTRs (AH008413.1), we conclude that K111 is the human variant of the K105 provirus. Furthermore, the K111 provirus clusters most closely with chimpanzee K105 in phylogenetic trees of gag, pol, and env, as well as chimp and human published K105 5' and 3' LTR sequences (data not shown). The 12q13.2 provirus was sequenced in this study (described below). Provirus sequences were deposited into GenBank (accession numbers: JN675007-JN675097), along with their respective flanking sequences (accession numbers: JN675098-JN675187).
Separate searches were performed using the UCSC Genome Browser to identify chromosomal coordinates of HML-2 solo LTRs. We queried the published sequence for elements corresponding to one of three HML-2 LTR subgroups: LTR5Hs (canonical sequence is ~986 bp); LTR5A (~1004 bp); or LTR5B (~1002 bp). Sequences corresponding to solo LTRs were extracted, aligned using ClustalW, and manually edited in BioEdit v.7.0.9.0 as described above. LTRs associated, and in the same orientation, with internal HML-2 gene sequences, were excluded from this analysis to ensure that only solo LTRs were analyzed. For the remaining elements, an arbitrary cut-off of 750 bp was used to include only the most intact elements per group.

For transfection, 5×10⁶ HEK-293T cells were plated 24 h prior to addition of a complex comprising plasmid DNA and Fugene 6 that facilitated DNA transport into the cells (as described by the manufacturer; Roche). The HIV type 1 (HIV-1) gag-pol construct pCMV-Δ8.91 (Zufferey et al., 1997 (link)) and green fluorescent protein (GFP) reporter construct pCSGW (pHR′SIN-cPPT-SGW, which incorporates the eGFP cassette driven by the U3 part of the spleen focus forming virus long-terminal repeat sequence; Demaison et al., 2002 (link)) or the firefly luciferase reporter construct pCSFLW (where the luciferase gene has been cloned into pCSGW in place of GFP; a kind gift from Dr B. Capecchi, Novartis Vaccines and Diagnostics, Siena, Italy), were transfected concurrently with the required envelope construct at a ratio of 1 : 1.5 : 1 μg, respectively. MLV gag-pol construct pCMVi (Towers et al., 2000 (link)) and GFP reporter construct pCNCG [a LNCX plasmid (CLONTECH) encoding enhanced GFP, with CMV driving expression of the RNA] or firefly luciferase reporter construct (Op De Beeck et al., 2004 (link)) were used at the same ratio as the HIV constructs. In each case, cells were washed 24 h post-transfection and incubated with fresh media. Supernatants were harvested 48 and 72 h post-transfection and titrated on HEK-293T, NP2, BHK-21, TE671 and N2A cell lines. The remaining virus was stored at −80 °C. Fresh or frozen pseudotype aliquots were used for virus titrations and neutralization assays, respectively. Each round of freeze-thaw resulted in an average loss in virus titre of 5.7 % for CVS-11, 3 % for EBLV-1, 5.3 % for EBLV-2 and 2% for VSV pseudotype (see Supplementary Fig. S1, available with the online version of this paper).

We acquired Raman spectra from spores using our custom-built LTRS instrument. The instrument is built around an inverted microscope (IX71, Olympus) [44 , 60 (link)]. We used a Gaussian laser beam operating at 785 nm (Cobolt 08-NLD) that is coupled into the microscope using a dichroic shortpass mirror with a cut-off wavelength of 650 nm (DMSP650, Thorlabs). Imaging and focusing of the beam were achieved by a 60 $\times$ water immersion objective (UPlanSApo60xWIR, Olympus) with a numerical aperture of 1.2 and a working distance of 0.28 mm. The same laser was used for Raman light excitation. In general, we operated the laser at a fixed output power of 100 mW corresponding to a power of about 60 mW in the sample (total energy of 1.2 J when exposed for 20 seconds). This power chosen was well below those previously recorded to damage spores [61 (link), 62 (link)].
We collected the backscattered light by the microscope objective and passed it through a notch filter (NF785-33, Thorlabs) to reduce the Rayleigh scattered laser line. Further, to increase the signal-to-noise ratio, we mounted a 150 $\mathrm{\mu }$ m diameter pinhole in the focal point of the telescope. The filtered light was coupled into our spectrometer (Model 207, McPherson) through a 150 $\mathrm{\mu }$ m wide entrance slit where a 600 grooves/mm holographic grating disperses the light [63 (link)]. The Raman spectrum was then captured using a Peltier cooled CCD detector (Newton 920N-BR-DDXW-RECR, Andor) operated at -95 ${}^{\circ }$ C. Our system has a Raman wavenumber spectral resolution of < 3 cm ${}^{-1}$ and accuracy of $\sim$ 3 cm ${}^{-1}$ .

We prepared a sample by placing a 1 cm diameter ring of 1 mm thick vacuum grease on a 24 mm $\times$ 60 mm glass coverslip. We added 5 $\mathrm{\mu }$ l of the spore suspension into the ring, after which we sealed it by placing a 23 mm $\times$ 23 mm glass coverslip on top. After the sample was placed in the LTRS instrument, we measured the Raman spectra of the spores using 2 accumulations of 10 seconds. We measured 20 individual spores for each sample (10 measurements in the 600-1400 cm ${}^{-1}$ and 10 in the 1000-1700 cm ${}^{-1}$ range), for 180 measurements in total. There were also triplicate controls at each spectral range. The background spectrum of the spore suspension was also measured and subtracted.