Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.

Schistosomiasis is a major neglected tropical disease (NTD), which afflicts > 200 million people in developing countries. The genome sequence of the schistosome parasite has been decoded; it includes > 10,000 genes. New approaches to control this NTD are sought and genomic information may provide targets for new treatments. Methods to determine the role and importance of specific genes would facilitate these tasks. The retrovirus HIV-1, the causative agent of HIV/AIDS, has been extensively studied and modified for use in biomedical research. Using a lab-modified form of HIV-1, we manipulated the genome of Schistosoma mansoni, one of the major species of schistosomes. Lab-modified HIV-1 infected schistosomes and inserted in the chromosomes of the parasite. These chromosomal insertions were mapped using next generation sequencing and were distributed throughout the chromosomes including the sex chromosomes. The findings were notable since they revealed that HIV-1 was active within cells of S. mansoni, and they provide the first demonstration that HIV-1 can integrate into the genome of an invertebrate. They pave a route forward for investigating new therapies for schistosomiasis.

There has been progress in developing functional genomics for schistosomes and some other flatworms [ 12 ]. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped murine leukemia virus (MLV) was shown to transduce schistosomes, integrating the provirus into the chromosomes of Schistosoma mansoni [ 15 , 16 ]. (Production of viruses with foreign viral envelope proteins is termed pseudotyping; pseudotyped is undertaken to increase host species and cell type tropism and/or enhance stability of the virions [ 17 ].) Germ-line transgenesis has been achieved by transducing S. mansoni eggs with VGV-G MLV, enabling the establishment of stable lines of transgenic parasites [ 16 , 18 ]. In addition, eggs also might be transducible by pseudotyped human immunodeficiency virus-1 (HIV-1) [ 19 ]. Lentiviruses such as HIV-1 possess a desirable attribute for functional genomics, since these viruses can infect both dividing and terminally differentiated non-dividing cells; MLV can infect the former but not the latter [ 20 , 21 ]. However, critical details are missing on the capacity of lentiviruses to integrate into chromosomes of flatworms and transcribe transgenes. In particular, given evolutionary divergence of flatworms and humans, the natural host HIV-1, it is necessary to characterize the preferred regions of integration before using lentiviral vectors for functional genomics. Here we report that infection of S. mansoni with pseudotyped HIV-1 lead to attachment of virions, reverse transcription of the RNA genome of HIV-1, and integrase-catalyzed insertion of the provirus into the genome of the blood fluke, and characterize the sites of integration. HIV-1-based manipulation of these parasites should enable advances in functional genomics for schistosomes and related platyhelminth pathogens.

Schistosomiasis is considered the most important helminthic disease of humanity in terms of morbidity and mortality, and is one of the major neglected tropical diseases (NTDs) [ 1 – 4 ]. Whereas > 90% of cases occur in Africa, where the major burden of disease lies, a recent outbreak in Corsica confirmed its re-emergence in Europe [ 5 ]. To accelerate discovery of intervention targets for schistosomiasis and to provide exploitable insights into the parasite biology and pathogenesis, concerted efforts are in train to produce reference genome sequences of the human schistosomes and related helminths [ 6 – 11 ]. To capitalize on the investment in flatworm poly-omics to identify novel control strategies, high-throughput systems for comprehensive studies of gene function have now become essential. However, because parasitic flatworms at large are difficult to maintain in the laboratory due to complex developmental cycles, they remain recalcitrant to genetic/cellular manipulations, presenting a significant bottleneck for adapting state-of-the-art approaches to elucidate gene function [ 12 ]. Current large-scale approaches, mainly involving medium-throughput RNAi screening [ 13 , 14 ], currently provide a veneer only of information on gene function since the knowledge of characteristics and regulation of specific gene expression remains limited. To profoundly probe function at scale, protocols for routine manipulation of the genome need to be established and optimized; genes need to be disrupted, transgenes inserted, and expressed in a sustainable, and even tunable, fashion.

Subsequently, numbers of integrations per worm were estimated. The WGS library was constructed using 1,700 ng of genomic DNA from ~5,000 schistosomula exposed to virions. Given the diploid genome of S. mansoni has an estimated mass of 0.79 picogram [ 6 ], the total number of nuclei in the WGS was ~2.2 x 10 6 . WGS was performed to a depth of sequence coverage of ~48 haploid genome-equivalents, resulting in 207 million paired (and properly mapped) reads among which 60 integrations were detected. Given that a diploid genome is equivalent to 7.29 million reads, the number of expected integrations per nucleus is 60 x 7.29/207 = 2.1 integrations (95% confidence intervals 1.6, 2.7), representing ~2,100 integrations per schistosomulum (it has been estimated that the 48 hour-old schistosomulum comprises ~1,000 nuclei [ 26 ]).

Given that both the qRAP and TraDIS approaches revealed that HIV-1 provirus had integrated widely into the schistosome genome, we undertook Whole Genome Sequencing (WGS) to precisely quantify integrated HIV-1 proviruses. Following WGS of genomic DNA from virion-exposed schistosomules to a depth of 48X coverage, Illumina reads were aligned to the genomes of both S. mansoni and HIV-1. Alignments were curated to remove false positive integrations and reads entirely of schistosome origin. Reads were assigned to mapping categories according to their position in either genome: 1) sequence reads containing HIV-1 adjacent to schistosome sequence, i.e. integration site within the read; 2) independent, i.e. pairs of sequence reads with one read aligned to the schistosome genome and the other to HIV-1; and 3) read pairs that matched only HIV-1, i.e. reads solely of lentivirus origin ( S5A Fig and S4 Table ). The WGS data revealed 82 reads among the three categories, and 60 integrations, 35 and 25 events within categories 1 and 2, respectively ( S5A Fig ). S5B Fig , presents a representative alignment of reads mapped to the genomes of S. mansoni and HIV-1, i.e. sequence reads containing partial HIV-1 sequence and partial schistosome sequence. This particular HIV-1 integration event, within the ZW chromosome, was one of 35 events in category 1 (above) ( S5 Fig ).

Panel A . Left, pie chart showing the percentages of the indicated regions in the genome of S. mansoni. Center, pie chart showing the percentages of HIV-1 integrations recovered from the 5’-LTR-end library classified according to the function of the mapping site. Right, pie chart showing the percentages of HIV-1 integrations from the 3’-LTR-end library classified according to the function of the mapping site. Panel B . Box plots showing expression of genes carrying integrated HIV-1 provirus (yellow) and transcriptomes at large (green) of several developmental stages. The lines extending parallel from the boxes (commonly referred as “whiskers”) indicate variability outside the upper and lower quartiles of the set of values. The upper and the lower limits of the whisker indicate the upper and lower extreme values, respectively. RPKM, reads per kilobase per million mapped reads.

Further analysis of the integration events revealed that exons contained 4% of the integrations, introns contained ~34.5%, whereas ~62% were detected within non-coding regions. By comparison, 4, 39 and 57% of the S. mansoni genome is composed of exons, introns, and non-coding regions, respectively ( Fig 5A ). Despite the apparent concordance of integration frequencies with genome composition of exons, introns and non-coding regions, statistical analysis revealed a significant tendency of proviral HIV-1 to integrate into non-coding regions (binomial proportion one-tailed test, P ≤ 0.01); this was dissimilar to MLV, which did not show bias for any particular region [ 16 ].

Panel A. Schematic region within chromosome 1 (Chr 1) of S. mansoni indicating the number of integration events in contiguous 100 kb sections along the chromosome (~65 MB). Integrations recovered from the 5’- and 3’-LTRs-end libraries are shown in blue and red, respectively. Arrowheads indicate HIV-1 integrase-catalyzed events (see Table 1 ). Panels B, C. Representative schistosome loci targeted by integrase-driven HIV-1 in chromosomes 2 and 5, respectively. Integrations from the 5’- and 3’ LTR-end are shown with blue or red triangles, respectively. Arrows indicate integrations into exons of protein-coding genes, i.e. Smp_146570.1 in chromosome 2 (Panel B) and Smp_061540.1 in chromosome 5 (Panel C). Genome coordinates of integration events indicated here were: chromosome 2 (11680000 to 11830000) 5’-LTR library: 11703778, 11734288 (exon 10 of Smp_146570), 11812331, 11812694, 3’-LTR library: 11682679, 11701658, 11721703, 11786909, chromosome 5 (8480974 to 8659352) 5’-LTR library: 8480974 (exon 6 of Sm_061540), 8580018, 8586055, 3’-LTR library: 8484589, 8529907, 8580599, 8594743, 8621686, 8632719, 8638819, 8659352. Schematics provided in panels B and C were obtained and modified from WormBase ParaSite, http://parasite.wormbase.org/ [ 83 ].

Proviruses of HIV-1 distributed throughout the eight pairs of chromosomes of S. mansoni. A frequency distribution of integration events along the entire ~65 Mb length of chromosome 1 (Chr 1) illustrated the rate of integrations throughout the nuclear genome as recovered using TraDIS. Some regions represented integration hotspots with a rate exceeding five integrations of HIV-1 provirus per 100 kb of chromosome. HIV-1 integrase-mediated events are indicated with arrowheads above and below the windows; here the dinucleotides CA and TG were characterized in the sequenced analysis at the termini of the integrated 3’-LTR and 5’-LTR of the provirus, respectively ( Fig 4A ), evidence of catalysis by HIV-1 integrase [ 25 ]. Examples of integration events within chromosomes 2 and 5, as detected in the 5’-end LTR and 3’-end LTR libraries, are shown in Fig 4B and 4C . The event characterized in chromosome 2 lies within exon 10 of Smp_146570, a histidyl-tRNA synthetase-related protein ( Fig 4B ), and that in chromosome 5 lies in exon 6 of Smp_061540, an amino acid transporter homologue ( Fig 4C ). The genes were inferred by protein orthology and coordinates of the integration events are provided ( Fig 4 legend). Note also numerous other integration sites, the positions of which are indicated with the arrowheads; blue colored arrowheads indicate events detected in the 5’-LTR-end library and red-colored in the 3’-LTR-end library ( Fig 4B and 4C ).

Panel A . Schematic of HIV provirus within the schistosome genome highlighting the termini of the 5’- and 3’-LTRs. Panels B, C . Representative integration boundaries (vertical red line) between the 3’-LTR- or 5’-LTR-ends of the provirus and schistosome genome within chromosome 1 and Z/W, respectively. The provirus dinucleotides CA at the 3’-LTR and TG at the 5’-LTR (CA in the reverse complementary to the 5’-LTR as indicated in Panel A) are highlighted. In the multiple sequence alignment, the top line is the sequence of the schistosome genome, the middle line is the 100 bp-sequence of the indicated TraDIS reads, and the bottom line shows the sequences at the termini of the 3’-LTR (B) or 5’-LTR (C) of HIV-1 clone pNL4-3 [ 69 ] employed to infect the schistosomes. Coordinates are indicated at the ends of the alignment.

Mapping the integrated proviruses to the schistosome reference genome revealed a broad distribution of integrations throughout all eight chromosomes of the parasite, comprising chromosomes 1 to 7 and Z/W, the sex chromosomes. Notably, integrations into the mitochondrial genome were also mapped ( Table 2 ). Similar findings were apparent from analysis of sequences mapped from either the 5’-LTR- or 3’-LTR-end libraries. It was evident that numerous integrations of HIV-1 provirus into the schistosome genome had been catalyzed by integrase, given the presence of the diagnostic dinucleotides CA at the 3’-LTR and TG at the 5’-LTR termini of HIV-1 at the integration junctions immediately flanking the schistosome genome ( Fig 3A and S1 Table ) ( S4 Fig ). Fig 3B and 3C present representative integration boundaries (vertical red bar) between the 3’-LTR termini of the provirus and the schistosome genome within the chromosome 1, or 5’-LTR-ends and chromosome Z/W, respectively. Sequences of a series of junctions of additional, representative integrations recovered using TraDIS from the 3’-LTR-end library are shown in S4 Fig .

In order to identify and map integrated HIV-1 proviruses within the reference genome of S. mansoni [ 6 ], an Illumina sequencing-based approach that utilized PCR to enrich for the integration events was adapted from a procedure named TraDIS (Transposon Directed Insertion-site Sequencing), which had been employed to characterize transposons in bacterial genomes [ 24 ]. The latter had been successfully adapted to localize integrations of VSVG-pseudotyped Murine Leukemia Retrovirus (MLV) in somatic and germ line-derived cells of schistosomes [ 16 ]. Illumina libraries were prepared from genomic DNA and then amplified to enrich for integration sites ( S1 and S2 Tables). High throughput sequencing of the TraDIS libraries constructed from both the 5- and 3’long terminal repeats (LTRs) of HIV-1 yielded >25,000 paired sequence reads with HIV-1 start sites from libraries constructed from both the 5’- and 3’-long terminal repeats (LTRs) of the lentivirus. About ~8,000 integrations were identified ( Table 1 ), comprising 1,827 and 6,258 non-redundant events from the 5’- and 3’-LTR libraries, respectively. Of these sites, most were unique clusters where neighboring integrations were separated by > 250 bp.

Thereafter, integration of HIV-1 cDNA into the schistosome genome was investigated. Integration of the provirus in human cells has been earlier assessed using a quantitative two-step Alu-based nested PCR [ 22 ]. We modified this approach to target multi-copy endogenous elements present in the schistosome genome; a method termed ‘quantitative Retrotransposon Anchored PCR’ (qRAP) [ 23 ]. Genomic DNA (gDNA) extracted from schistosomula was subjected to nested PCR employing a primer specific for the gag gene of HIV-1 in tandem with primers specific for several endogenous retrotransposons known from the genome of S. mansoni ( Fig 2C ). The relative copy number of integrated HIV-1 as estimated by qRAP was significantly higher in schistosomes transduced with active virions compared to the negligible signals from parasites exposed to heat-inactivated virions, at both 24 and 48 hours after transduction ( Fig 2D and 2E ). These findings indicated that HIV-1 cDNA reached the nuclei of schistosomes, and that the proviruses integrated into the genome of the parasite, at least in regions proximal to the endogenous retrotransposons employed as anchors for the qRAP [ 22 ]. Curiously, two inhibitors of reverse transcriptase, azidothymidine and nevirapine, each with a discrete mode of action, and an inhibitor of integrase, the diketo acid derivative 118-D-24, failed to block these events, as determined by qRAP targeting integration events ( S3 Fig ).

Panel A . Quantitation of negative-strand, strong-stop HIV-1 cDNA in genomic DNA of schistosomula 24 hours after exposure to intact or heat-inactivated virions. Panel B . Quantitation of the late, positive-strand HIV-1 cDNA in schistosomula at 24 and 48 hours after incubation with virions. Panel C. Schematic representation of the nested two-step, quantitative retrotransposon-anchored PCR (qRAP) for relative quantitation of HIV-1 provirus integrated into the schistosome genome. Products of the first reaction using retrotransposon-specific primers were subjected to secondary PCR using provirus-specific primers. Panel D . Detection of HIV-1 provirus integrated into the schistosome genome using the primer set no. 1 specific for retrotransposons SR1 and SR2. Panel E . Detection of HIV-1 provirus detected with primer set no. 2, specific for fugitive, SMα, and Boudicca. Statistical analysis: Student’s t-test; *, **—P ≤ 0.05, P ≤ 0.01 (active vs. heat-inactivated virions). The experiments were repeated ≥ three times.

Quantitative PCR (qPCR) of DNA extracted from schistosomula exposed to active or heat-inactivated virus was performed employing HIV-1 specific primers to estimate the copy number of HIV-1 cDNA molecules. Both early, strong-stop and late, positive-strand HIV- specific cDNAs were detected in parasites exposed to active HIV-1, whereas few copies were detected in parasites exposed to heat-inactivated virions ( Fig 2A [early strong-stop; P ≤ 0.05, Student’s t-test] and 2B [late, positive-strand; P ≤ 0.01]). These findings established that reverse transcription of the RNA genome of HIV-1 had proceeded in the cells of virion-exposed parasites.

Panels A, B, C, schistosomula exposed to culture medium containing 8 μg/ml polybrene for three hours, fixed and probed with anti-VSV-G rabbit polyclonal antibody and secondary Alexa Flour 488 chicken anti-rabbit IgG (control). Panels D, E, F, schistosomula exposed to VSV-G-pseudotyped HIV-1 in culture medium containing 8 μg/ml polybrene for three hours, fixed and probed as described in A-C. Micrographs captured with same exposure and magnification 40X and 63X; Red arrows indicate areas of high Alexa Flour 488 fluorescence within the schistosomulum tegument. Panels G, H, Gray value profiles of the cross-sections of images of the virus-exposed (H) and non-exposed (G) schistosomules. Red arrows indicate peaks of Alexa Flour 488 fluorescence on the tegument of the worm. Immunolocalization assays were undertaken at least twice for schistosomula and adult schistosomes.

The successful attachment of VSV-G pseudotyped HIV-1 to the tegument of schistosomes was demonstrated using an antibody specific for VSV-G. Specific binding was observed to the surface of both schistosomula ( Fig 1A–1F ) and adult worms ( S1 and S2 Figs) following exposure to the virions. An evident fluorescence signal emitted by Alexa Fluor 488-labeled anti-VSV-G antibody was detected and measured using spinning disk confocal microscopy ( Fig 1G and 1H ). Moreover, the signal intensity observed mainly in the surface of the virion-transduced parasites significantly increased over three hours exposure ( S2 Fig ). These results demonstrated time-dependent attachment of the virions to the schistosome tegument. In addition, the binding pattern seen on the tegument of both the schistosomules and adult worms revealed a focal rather than general binding to the surface of this developmental stage ( 1D, 1E , S1E and S1F Figs). Schistosomes not exposed to virions and incubated with VSV-G primary antibody and schistosomes exposed to virions and incubated with the secondary antibody only did not exhibit fluorescence, thereby indicating specific binding by both the primary and secondary antibodies. Although, autofluorescence was evident in schistosomules and adult worms ( Fig 1A and 1B and S1D Fig ), that ‘fluorescence’ pattern was distinct and readily distinguished from the Alexa Fluor 488. signal ( Fig 1H ).

Discussion

This report characterizes the integration of HIV-1 provirus within the chromosomes of Schistosoma mansoni. It also provides, to our knowledge, the first demonstration of integration of HIV-1 into the genome of an invertebrate. Inoculation of cultures of schistosomes with HIV-1 led to attachment of virions to the surface of the blood fluke, reverse transcription of the RNA genome of HIV-1, and integration of the provirus into the genome. Notwithstanding that transduction of these parasites was facilitated by pseudotyping virions with VSV-G, and that transduction proceeded in the absence of CD4 and other cellular receptors expressed on activated T and other receptive cells, biochemical processes that evolved for parasitism of primates [27] by lentiviruses proceeded within the cytoplasm and nuclei of schistosome cells, including reverse transcription of the RNA genome, assembly of a pre-integration complex, transit to the chromatin and integrase-catalyzed integration of the provirus into the chromosomes.

Lentiviruses traverse the nuclear envelope, and consequently HIV-1 can productively transduce non-dividing target cells, such as macrophages. By contrast, gammaretroviruses such as MLV cannot transverse the nuclear envelope; rather, MLV accesses the chromosomes at mitosis following dissolution of the nuclear membrane [28, 29]. HIV-1 possesses a conclusive advantage over MLV for functional genomics since it can infect both dividing (as can MLV) and terminally differentiated non-dividing cells (which MLV cannot) [20, 21]. For example, HIV can transduce non-dividing human cells, including stem cells, prior to differentiation, and terminally differentiated cells, such as monocyte-derived macrophages, astrocytes and microglia [30, 31]. The pre-integration events in the developmental cycle of HIV are essentially discrete from the events of the MLV life cycle: the pre-integration complex (PIC) of HIV mixes with chromatin after transit through the intact nuclear envelope whereas the MLV PIC mixes with chromatin during mitosis [32]. Whether the PIC of HIV can integrate in the chromosomes without passage through the nuclear envelope remains to be determined but this seems unlikely given the dissimilarity in structure of the PICs of HIV and MLV. It is feasible that HIV-1 entered the nuclei of S. mansoni cells during interphase, and integrated into non-dividing cells [33]. HIV-1 also may access the chromosomes at mitosis although evidence that this mode of replication leads to productive infection is not yet available. Nonetheless, one or both of these routes may have led to the widespread integration of HIV-1 in the schistosome genome.

In view of earlier demonstrations of transduction of schistosomes by MLV [15, 16] and the present findings with HIV-1, it is clear now that pseudotyped retroviruses can accomplish chromosomal integration and vertical transmission in schistosomes. Moreover, infection by HIV-1 appears to be efficient: based on titers of virions to which these parasites were exposed and, assuming the presence of ~104 virion particles in one pg/ml of p24CA (the major structural protein of the HIV-1 virion capsid; there are ~1,000 p24CA proteins in the mature virion) [34], each schistosomulum was exposed to ~0.5 x 106–1x106 virions. Since the WGS analysis detected ~2,100 integrations per schistosomulum, we estimated an integration efficiency of ~0.25–0.5%. Spinoculation, centrifugation of the worms in the presence of virions [developed for human T cells [22, 35], delivered >5-fold increase of viral entry and increased numbers of integrations (S6 Fig). It should be noted that natural infection of schistosomes by HIV-1 in people co-infected with both pathogens is highly unlikely to occur given that schistosomes do not express HIV-1 receptors. In this study, the HIV-1 virions were pseudotyped with G protein of vesicular stomatitis virus (VSV-G), which binds with a highly ubiquitous LDL family receptors (LDLR) and endows the pseudotyped virus with pantropism [36]. This process does not happen in natural conditions. This is supported by the lack of HIV-1 sequences in the curated genome of S. mansoni [6, 8], which represents an established laboratory strain of S. mansoni that is maintained in rodents [37].

Given the mechanism of productive infection by HIV-1 of CD4+ T lymphocytes, events in schistosomes would have taken place in concert with cellular factors. Some processes, including endogenous reverse transcription, can proceed in vitro outside host cells [38–40], and perhaps proceeded here in the presence of a minimal number of human cellular factors incorporated into the virion during virion production. However, chromosomal integration and other processes require specific factors in human cells, suggesting that orthologues or even less conserved surrogates from schistosome were adequate. Homologues of factors that contribute to establishment of infection appear to be present in S. mansoni (S5 Table), including a capsid-binding protein (Smp_094810), reverse transcription complexes-interacting protein (Smp_100090), importins involved in the nuclear translocation of the viral pre-integration complex e.g. Smp_051210, Smp_142770, and integrase-interacting protein (Smp_125050). However, HIV-1 exhibited divergent integration preference in schistosomes compared to T cells, a site preference influenced by lens-epithelium-derived growth factor, LEDGF/p75. LEDGF/p75 stabilizes and tethers the intasome, a tetramer of integrase proteins bridging the termini of the provirus, to the nucleosome [41, 42]. Absence of an orthologue of LEDGF/p75 may account for discrete integrations profiles between schistosome and human cells.

Integrase-dependent integration of full-length proviruses took place; the intact termini of the LTRs provided cogent evidence of catalysis by integrase [43]. Recovery of twice as many events from the 3’-LTR library suggested that mutations of the provirus also occurred such that rearrangements, truncations or deletions of the HIV-1 proviral genome influenced the efficiency of the TraDIS targeting the 5’-LTR. These phenomena occur during HIV-1 infection [44–46], but may be less surprising in the exotic setting of tissues of a schistosome. Truncated versions of MLV occur in schistosome chromosomes [15, 16]. Variants of truncated HIV-1 genomes and integration junctions that lacked CA residues at the termini of the HIV-1 provirus suggested integrase-independent recombination of incomplete reverse transcripts. These integrations may have resulted from homologous recombination or from non-homologous end joining [47, 48]. The entire lentiviral cDNA containing the central flap, a plus strand overlap in the 3’-terminus of pol, has an advantage over truncated cDNAs for importion into the nucleus [49, 50]. Yet completion of reverse transcription did not appear to be essential for insertion of viral cDNA into the schistosome genome. Perhaps HIV-1 exploited an alternative nuclear import mechanism in schistosomes. Numerous integrations of 3’-end HIV-1 cDNAs, which may represent intermediate products of reverse transcription, supported this possibility. In like fashion, truncated forms of integrated HIV-1 provirus that appear to represent products of incomplete reverse transcription frequently occur during HIV-1 infection in humans [51]. Since spinoculation enhanced contact of virions with the surface of the schistosome, the virions may have transduced the tegument rather than deeper tissues. The tegument of blood stream forms of the schistosome is syncytial, with multiple nuclei that are not in cell cycle synchrony [52, 53]. This arrangement may have facilitated contact of provirus with chromosomes, and recombination of incomplete reverse transcripts of HIV-1 into the genome.

Integrated proviruses preferred less gene rich regions but nonetheless proviruses were distributed in both coding and non-coding regions in the genome of S. mansoni. Within the Retroviridae, species-specific preferences for sites of integration have evolved so that, for example, HIV-1 prefers actively transcribed genes in euchromatin whereas MLV exhibits bias for promoters of actively transcribed genes, enhancers and CpG islands [54]. Indeed, half the MLV integrations occur within < 2% of the genome of some human cell lines [55]. By contrast, the distribution of HIV-1 integrations in schistosomes was reminiscent of site selection in human chromosomes by prototype foamy virus, a spumavirus, and the Alphavirus avian sarcoma leukosis virus (ASLV) [56]. ASLV prefers open euchromatin but does not show bias for transcriptional elements [57]. Other differences were seen including the inactivity of inhibitors of reverse transcriptase and integrase in schistosomes and integration into the mitochondrial genome. The inactivity of integrase inhibitor 118-D-24 on HIV-1 integration seemed consistent with the finding that numerous integrations may have occurred independent of insertion by integrase. However, the same or higher efficiency of reverse transcription in the worms treated with azidothymidine and nevirapine, suggested distinctive mechanisms of elimination of all these compounds, the inhibitors of reverse transcription and integrase, from schistosome cells. This might have been accomplished by aquaporins and other transporters active in schistosomes [58, 59]. Moreover, the inhibitors may not have entered schistosome cells that had been infected with HIV-1. Integration into the mitochondrial genome was notable given that this phenomenon may not have been reported in HIV-1 infected human tissues, as indicated by absence of reports of this type of event within the Retrovirus Integration Database, a public database for retroviral insertion sites [60].

A compelling attribute of HIV-1 versus gammaretroviruses such as MLV is the ability of the former to efficiently infect both dividing and terminally differentiated cells. Here we tested the HIV-1 transduction in the schistosomula and adult worms where many cells are differentiated and do not proliferate. Since we sought to investigate reverse transcription and chromosomal integration, access to substantial quantities of RNA and DNA from the parasites facilitated these analyses. Therefore, it was less challenging to investigate these biochemical processes of HIV-1 in blood stage schistosomes, schistosomula and adults (rather than eggs). In future studies we plan to investigate the feasibility of deriving transgenic lines of schistosomes by transducing eggs with VSVG-HIV-1, given that eggs of schistosomes have been successfully transduced with pseudotyped MLV [61]. Eggs of S. mansoni have been exposed also to pseudotyped HIV-1 virions carrying transgenes encoding microRNA-adapted short hairpin RNAs targeting genes expressed in schistosome eggs. The HIV-exposed eggs were inoculated into the pulmonary circulation of mice, which led to phenotypic changes in the inflammatory response, presumably following gene knockdown [19].

Establishment of transgenic lines of schistosomes, derived from retrovirus- and lentivirus-transduced eggs, expressing transgenes including Cas9 nuclease, should now be achievable. This would enable generation of specific knock-out lines using CRISPR-Cas9 gene editing as recently demonstrated for a parasitic nematode [62] and induction of stable gene knock-down from expression of shRNAs from integrated transgenes delivered using lentiviral vectors [14, 19, 63]. The performance of cis-regulatory elements to drive the expression of transgenes, insulator elements to prevent chromatin silencing position effects, and the use of selection markers need to be further investigated. How the widespread integration of HIV-1 in the schistosome chromosomes influences gene expression awaits investigation, as does the impact of the integration bias. The high-throughput approaches employed here to estimate the number of integration events investigated genomic DNAs pooled from large numbers of schistosomules, and hence the relatively high number of integrations represents what took place within the population of pooled HIV-transduced parasites. Transducing schistosomes with high titers of HIV-1 may also facilitate insertional mutagenesis-based forward genetics. Manipulation by transgenesis, knockout and/or gene editing by CRISPR/Cas 9-related approaches [64, 65] can be predicted to enhance understanding of these pathogens, their somatic stem cells [66], reproduction, longevity in infected hosts [10, 67], and intervention targets. These approaches may also facilitate establishment of sex-biasing gene drives to block the spread of schistosomiasis [68].

To summarize and conclude, this report presents enigmatic findings that reveal that certain steps of HIV-1 replication are active within cells of the human blood fluke, Schistosoma mansoni, a parasitic flatworm responsible for the major neglected tropical disease (NTD) schistosomiasis. Facile manipulation of schistosomes using lentiviruses should enable advances in functional genomics in these and related NTD pathogens including tapeworms, in particular concerning their non-dividing cells. Such approaches have hitherto been unavailable, and the lack of these kinds of tools underpins why the NTDs are neglected: the helminth NTD pathogens have not been readily tractable to laboratory investigation. Unlike the retrovirus MLV, which we investigated previously [15, 63], HIV-1 integrates into non-dividing cells. This represents a distinct advantage in applications such as transient transformation of adult and schistosomula stages, and thus is a substantial advancement in the functional genomics of these important parasites. A corollary of the findings is that lentiviral pre-integration complexes exploit either evolutionary conserved mechanisms or that HIV-1 can employ diverse strategies of nuclear import and integration. Although HIV-1 has been considered as a specialist virus because it uses species-specific receptors for host cell entry, the new findings suggest, rather, that it is generalist in use of intracellular pathways at post-entry steps of infection.