Analysis of PERV-C superinfection resistance using HA-tagged viruses

Background Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect. Results We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed. Conclusions The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx. Supplementary Information The online version contains supplementary material available at 10.1186/s12977-023-00630-x.

Generally, retroviruses can create SIR in cells by establishing an interference mechanism after the initial (primary) infection that prevents infected cells from being superinfected by a similar or same type of virus [37].SIR is likely to result from receptors being hidden or downregulated after interaction with the known virus's glycoprotein, either intracellularly or at the cellular surface [37][38][39][40].Retroviruses like murine leukemia virus (MuLV), human immunodeficiency virus (HIV) and foamy virus (FV) use Env-mediated SIR mechanisms, so a similar mechanism may be assumed for PERV [36,37].It was shown that proviral gene products can mediate the restriction of viral replication [37].In consequence, targeted expression of virus specific genes should be considered as a potential protection mechanism against retroviral infections.Several examples have been published.
In 1975, Suzuki discovered the Fv4 gene, which encodes a retrovirus-related Env polyprotein from the Fv4 locus containing the mid pol-region, env and the 3′-long terminal repeat (LTR) [41][42][43].In vitro data of NIH 3T3 cell lines stably transfected with the cloned Fv-4 env gene confirmed SIR to ecotropic MuLVs [44].In vivo studies using transgenic mice carrying the Fv4 gene affirmed this result.The resistance correlated with the Fv-4 env expression level.It was assumed that the MuLV receptor is probably masked by the Fv4-Env complex [45].
For PERV, an evidence for PERV SIR mechanism was shown for reverse transcriptase (RT) defective PERV-A and -B released from a porcine kidney cell line (PK15 clone 15 cells) that had been inactivated by CRISPR/Cas9 [34,36].These cells still produce complete but dysfunctional virus particles, which were irregularly shaped and apparently immature [36].
The cells could not be reinfected and remained RT negative.However, the question if foreign PERV-A, -B, -C or -A/C was able to enter the cells or not was not sufficiently answered since all viruses used for this analysis were untagged wildtype viruses and nearly indistinguishable from the receding viruses.
In this study, we focused on PERV-C(5683), as a reference virus.We generated, for the first time, HA-tagged PERV-C viruses, alongside providing a blueprint for PERV tagging.Viruses have been tested for functionality and SI in an in vitro assay on primary infected and noninfected porcine ST-IOWA cells.The results demonstrate that ST-IOWA cells primarily infected with functional PERV-C(5683) subsequently show resistance to infection with the same type of virus.The resistance probably will be conveyed during the early phase of infection, as there was no integration to the host genome.Our data prove that SIR also exists for PERV-C and that SIR is mediated by functional as well as dysfunctional PERV particles.A finding that opens up new strategies while using viral components for cellular resistance [34][35][36][46][47][48].In addition, the tagged virus is a novel tool and data are suitable for the generation of e.g.PERV-A, -B and -A/C clones to further analyze PERV infection mechanism, perhaps in vivo.
The results show that the insertion of an HA-tag close to the signal peptide (SP-HA) or to the membrane anchor (RPep-HA) had no effect on virus functionality.An introduction N-terminal to the VRA region restrained viral replication.Due to the lack of vRNA expression, HA-VRA was examined for its ability to integrate into the host genome as a provirus.To this end, genomic DNA (gDNA) from ST-IOWA cells transfected with HA-VRA (21 days p.t.) was analyzed for HA-VRA integration by PCR using envCspecific primers.No integration of HA-VRA provirus into the host genome was detected (data not shown).Therefore, HA-VRA was excluded from further analysis.
Recombinant SP-HA and RPep-HA were analyzed for stable HA-tag integration and protein expression by immunofluorescence (IF) experiments and western blot (WB) analysis 56 days p.t. (Fig. 3).IF showed the presence of EnvC-HA proteins in transfected cell lines ST-IOWA/ SP-HA and ST-IOWA/RPep-HA (Fig. 3A).
WB analysis using an anti-HA-tag antibody confirmed that EnvC-HA is present as a precursor and in its processed forms namely the surface unit (SU) and the transmembrane unit (TM) (Fig. 3B).Depending on the HA-tag position, the detection of the cleaved variant is either N-terminal restricted to SU or C-terminal to TM.As expected, SP-HA (lane 2) showed one large protein band at approximately 70 kDa representing the precursor EnvC-HA (72.1 kDa).The precursor and the processed form SP-HA-SU (52.3 kDa) could not be differentiated.For RPep-HA, the precursor EnvC-HA (72.1 kDa) as well as TM-HA (20.9 kDa) were detected.
Electron microscopy (EM) was used to demonstrate that the integration of an HA-tag did not change the morphology of the virus particles (Fig. 4).EM of SP-HA and RPep-HA revealed a characteristic morphology of viral particles, showing a dense, collapsed core surrounded by evenly rounded capsid that is comparable to native PERV-C(5683).EM of ST-IOWA cells did not show any virus or virus-like particles (data not shown).
In continuation, the functionality of the PERV-C-HA viruses SP-HA and RPep-HA was demonstrated by incubation of native ST-IOWA cells with virus containing supernatant (SN) derived from the transfected cell lines (Table 2).Infection of ST-IOWA cells with SN from ST-IOWA/PERV-C(5683) cells served as positive control.The infection progress was monitored weekly, by quantification of PERV-C env and PERV pol vRNA expression, RT activity measurement and IF (Fig. 5).Irrespective of the PERV-C variant used for infection, vRNA expression was observed from the second week on reaching levels between 10 5 -10 6 copies/µL (Fig. 5A, B) and RT activities between 15 and 50 mU/mL were detected from the third week on (Fig. 5C).In addition, infected and non-infected cells were distinguished by IF at day 56 days post infection (p.i.) using an anti-HA-tag antibody and anti-EnvC peptide directed antiserum (Fig. 5D).

AlphaFold2 based structural analysis of HA-tagged PERV-C
PERV-C(5683) Env as well as the three HA-tagged EnvC were structurally analyzed and compared using Alpha-Fold2 software [49] to gain insight into possible changes in the PERV-C Env structure that resulted in functional or non-functional viruses.
Here, the predicted structures of the native EnvC and the labeled EnvC-HA variants were compared by a MUSCLE sequence alignment (Fig. 6).As a computational prediction that has to be justified, it was shown that the integration of the HA-tag led to slight conformational changes while comparing the rank 001, model 3 prediction.
In consideration of the pLDDT score (< 50) minor structural changes of SP-HA and RPep-HA like the slight conformational displacement of the SP and the proline rich region (PRR) are not taken into account.Interestingly both constructs showed a small conversion of the TM (p15E) with a pLDDT score between 40 and 90 when compared to the wildtype (Fig. 6AI, AII, CI, CII).For the non-functional variant HA-VRA, structural prediction revealed an almost identical structure of the SP (AA 1-43) and TM (AA 473-623) when compared with PERV-C(5683).However, the introduction upstream of the VRA resulted in an intrinsically twisted loop formed by the HA-tag (AA 86-94) (pLDDT score < 50).In addition, the PRR (AA 247-285) underwent a major structural change (pLDDT score < 50).The PRR of PERV-C(5683) consists of a broad loop region, whereas the alteration of the HA-VRA PRR resulted in a twisted, thus compressed loop placed at a different angle (Fig. 6 B I, B II). Alignment of the PERV-C-HA variants (Additional file 2: Fig. S2) showed that similar changes were observed for SP-HA and RPep-HA, whereas HA-VRA varies more from the others.While only minor differences were found in the TM (Additional file 2: Fig. S2 A), significant divergences were observed in the SP (Additional file 2: Fig. S2B) and PRR (Additional file 2: Fig. S2C).

Infection assay of ST-IOWA/(PERV-C(5683) cells
For the analysis of PERV-C SIR, virus producing ST-IOWA/PERV-C(5683) cells were challenged with PERV-C(5683)-HA viruses (SP-HA or RPep-HA) to set up superinfection (SI).The infection was followed for 56 days.Analysis was performed by qRT-PCR (Fig. 7A-C), RT activity assays (Fig. 7D), IF (Fig. 8A) as well as WB analysis (Fig. 8B).As a control for virus infectivity, i.e. functionality, native ST-IOWA cells were successfully infected with the same inoculum.For experimental readouts, PERV envC vRNA expression was quantified by qRT-PCR (Fig. 7A).Since SI cells and primary infected cells both express PERV-C env either with or without the HA-tag, levels of PERV-C env vRNA expression remained stable (10 6 -10 7 copies/µL).Primary and secondary infections were distinguished by a subsequent qRT-PCR using envC-HA specific oligonucleotides (Table 3).The results show no expression in SI cells when compared to the controls.For primary infected ST-IOWA control cells envC-HA vRNA was 10 5 -10 6 copies/µL and for secondary infected SI ST-IOWA/PERV-C(5683) cells it was 10 2 -10 3 copies/µL (Fig. 7B, C).RT activity assays were used to monitor retroviral enzyme activity of the infected cells showing that all cells were RT positive (Fig. 7D).The presence of EnvC and EnvC-HA protein expression was analyzed by IF (Fig. 8A) and WB analysis (Fig. 8B) using a combination of PERV-C Env peptide and HA-specific antibodies.PERV-C Env proteins were detected in all cells except native ST-IOWA (ctr−) cells.HA proteins were only detected in the primary infected controls but not in the SI cells.Data confirmed that tagged viruses have the capacity to infect native ST-IOWA cells, however, they did not superinfect primary infected ST-IOWA/PERV-C(5683) cells.
In addition, viral integration into the host genome of SI cells was excluded by PCR using PERV-C env and envC-HA specific primers (Table 3), analyzing gDNA of infected and SI cells (Additional file 4: Fig. S4).There was no HA-tagged proviral PERV-DNA detectable after superinfection.

Discussion
Multiple proviral copies of PERV are spread throughout the pig genome.Some of them encode functional PERV of which only PERV-A, -B and -A/C are able to infect human cells in vitro.They are considered as a risk in the course of XTx.Up to now, there is no documented in vivo transmission of PERV except the effect of microchimerism after transplantation of porcine tissues and cells [50,51].However, the lack of evidence that polytropic PERV have no infectious potential for human xenotransplant recipients remains as a hurdle.Consequently, the generation of donor animals free of functional PERV and their protection from reinfection is a requirement for the development of porcine cellbased therapeutics.One approach was the genomewide inactivation of PERV using CRISPR/Cas9 [34].
A beneficial side effect was that inactivated PK15 cells still producing inactive viral particles maintain cellular resistance to SI.This suggests a potential protective effect after inactivation of PERV RT and provided a first insight how to benefit from PERV SIR [36].
To carry on, we generated HA-tagged PERV-C(5683) as a model virus to inoculate PERV-C(5683) positive ST-IOWA cells in vitro, and confirmed that SIR is also valid for PERV-C.To this end, PERV-C(5683) viruses were labeled with an HA sequence tag located at three different regions of envC (SP-HA, HA-VRA and RPep-HA).SP-HA and RPep-HA viruses showed infectivity comparable to PERV-C(5683).In contrast, the integration of an HA-tag N-terminal to the VRA region resulted in an impaired viral infectivity.This finding indicates the structural importance of this region that was impeded e.g. by disrupting a binding motive or interfering with the protein structure while integrating new amino acids.Evidence that structural changes in HA-VRA lead to a loss of recombination capacity has been strengthened by computational analysis using AlphaFold2.The major changes are located in a loop region with limited prediction accuracy (pLDDT score < 50).Nevertheless, it is worth mentioning as they differentiate the nonfunctional variant HA-VRA from PERV-C(5683) and the functional variants SP-HA and RPep-HA.The responsiveness to insertion mutagenesis marks the VRA upstream region as a potential target for further virus inactivation studies, and might be applicable to other PERV classes.
The PERV HA-tagging allows a differentiation of primary and secondary infection as shown for PERV-C positive ST-IOWA cells.Thus, PERV-C SIR was confirmed.In addition to this study, HA-tagged PERV-C provides a useful tool to further study host-pathogen interactions, in vitro and in vivo.The positions that have been proven suitable for tag introduction in PERV-C are applicable for other PERV classes e.g.PERV-A, -B, -A/C.
In terms of SI, the aspect that ST-IOWA/PERV-C(5683) cells challenged by the same type of virus remain unaffected, raises questions regarding the mechanism.Either the presence of viral proteins, virus particle formation or the release of functional viruses itself seems to prevent the host-cell from new infection by an identical or similar virus.For PERV-C, it was proven that an integration of the tagged virus into the host genome did not occur.Even if SI cells were negative for envC-HA DNA, the entry of viral RNA into the host cell cannot be excluded.The inability to integrate could be caused by inadequate cell entry e.g.attachment, receptor binding or fusion or due to an insufficient post-entry affecting the uncoating or the integration itself.Data from MuLV and FV studies [41,45,52] suggest that retroviral SI is blocked at an early stage, e.g. by masking or downregulating the receptor through Env-binding or by a general upregulation of antiviral factors.For PERV, an analogous mechanism is expected.In PK15 and ST-IOWA cells the porcine PERV-A receptor (POPAR) is expressed on the RNA level and ST-IOWA cells show a basal expression of PERV-A env RNA [36].PERV-RNA expression is not sufficient to restrict SI, which indicates an inhibition on the protein level.
Consequently, the usage of PERV proteins as a protection against exogenous PERV infection is obvious.Takeuchi et al., 1998, demonstrated in their host range and interference studies that cells that actively produce e.g.PERV-A or -B are not susceptible to pseudotype virus of the same class [6].Similar results were shown by Argaw et al. [53].Infection with PERV excludes reinfection with PERV of the same class.Infection with different PERV classes is possible due to different host receptors.Since the tropism of PERV is determined by Env protein, it is the most likely factor to mediate SIR.Even if non-Env-mediated SIR mechanisms are described e.g.Gag/ MuLV [54,55] it seems unlikely for PERV.Gag-mediated SIR would be expected to protect against all PERV classes as it is highly conserved.Its mode of action could be that of a cofactor or carrier protein.
Eventually, SIR prevents amplification of PERV in the host genome through reinfection.On the other hand, ERVs including PERVs can amplify through intracellular retrotransposition [56,57], by transcription and subsequent reverse transcription leading to ERV sequences that are missing distinct parts of the LTR.Last but not least, in silico screening showed that env gene loss is associated with a significant (up to 30-fold) increase in genome copy number, implying that such env-less ERVs can be considered genomic superspreaders [58].This could be an explanation for multiple dysfunctional PERV copies in the pig genomes.
The newly generated PERV-C(5683)-HA viruses are a suitable tool for gaining knowledge on PERV-C hostpathogen interaction.An application of already existing cellular defense mechanisms against PERV infection on  the basis of SIR is a novel approach that will contribute to increase the virus safety of xenotransplants.

Conclusion
Elimination of functional PERV in XTx must be ensured.
In recent years, great success in this field has been achieved through the selection of PERV-C-free pigs and the genome-wide inactivation of PERV using CRISPR/ Cas9.However, preventing reinfection, i.e.SI, of donor animals with PERV is of importance.Therefore, we have developed HA-tagged functional PERV confirming that the mechanisms of SIR also applies to PERV-C.It opens up a new possibility using a natural existing protective mechanism against PERV, thereby supporting virus safety of XTx.

Design and preparation of PERV-C-HA plasmids
Design of HA-tagged PERV-C was performed starting with the viral backbone of PERV-C(5683) (KY352351.2) [26] cloned in pBluescript SK (+) (Agilent Technologies, Germany).EnvC of PERV-C(5683) was structurally analyzed using the open source tool PSIPRED [59] to highlight sections of amino acids that do not bear structural motifs (Additional file 1: Fig. S1A).Only sequence sections defined as coil were selected for integration of the tag.Integration into helixes or ß-strands was avoided.
Based on sequence alignments, structural analysis and on published functional studies [11,26,53,60] the HA-tag was introduced at AA 44-52, AA 86-94 or AA 639-647 resulting in SP-HA, HA-VRA and RPep-HA, respectively.Corresponding sequences are shown in Additional file 1: S1B-D.Integration of the HA-tag into PERV-C(5683) was performed by site directed mutagenesis using the Q5 ® Site-Directed Mutagenesis Kit (New England Biolabs, Germany) following the manufacturer's instruction.Primers used are shown in Table 1.

Cell lines and viruses
PERV-C was harvested from producer cell line ST-IOWA, carrying the molecular clone PERV-C(5683), KY352351.2.Native ST-IOWA cells [9,61] were used as negative control.These cells are susceptible to PERV-C infection and are RT negative as they are free of functional PERV-A and -B [62].Cells were cultured as described previously [26,63].Cells and corresponding RT activities of virus-containing SNs used for infection experiments are listed in Table 2.

In vitro transfection of ST-IOWA with PERV-C
For in vitro transfection, PERV-C DNA was purified by the EndoFree Plasmid Maxi Kit (Qiagen, Germany) as recommended by the manufacturer.ST-IOWA cells were seeded in triplicates of 2 × 10 5 ST-IOWA cells/well in sixwell culture plates (Sarstedt, Germany) 24 h prior transfection.Transfection was performed using the FuGene ® HD transfection reagent (Promega, USA) according to the manufacturer's instructions with a ratio of transfection reagent to DNA of 3:1.

In vitro infection of ST-IOWA and ST-IOWA/PERV-C(5683)
For in vitro infection, triplicates of 2 × 10 5 ST-IOWA cells/well, were seeded in six-well culture plates (Sarstedt, Germany) 24 h prior infection.Cells were incubated with 1 mL/well cell-free SN containing replication-competent PERV-C in different amounts according to RT activity values (Table 2) for 24 h.After washing the cells three times with 2 mL/well phosphate buffered saline (PBS) to remove input virus, cells were further cultivated for 56 days.Quantification of PERV-C expression and RT activity took place weekly from cell-free SN of pooled triplicates.Cell-free SN aliquots were stored at -80 °C until testing.

Preparation of viral RNA and quantification of viral RNA expression
Isolation of viral RNA from cell-free SNs was performed using the QIAamp ® Viral RNA Mini Kit (Qiagen, Germany), according to manufacturer's instruction.Onestep qRT-PCR was performed on PERV pol, env-C and env-C-HA in a LightCycler ® 480 (Roche Life Science, Swiss) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany) as recommended by the manufacturer.Detection and quantification of PERV-C env and pol vRNA was performed as described before, using primer pair EnvC-for2/EnvC-R3_AWG or F/R [36,63].For the quantification of env-C-HA, cycling conditions were as follows: RT-reaction (50 °C, 30 min); denaturation (95 °C, 15 min); 40 cycles of amplification (94 °C, 15 s, denaturation; 68 °C, 30 s, annealing; and 72 °C, 30 s, elongation).Melting curve analysis was performed as follows: 95 °C, 10 s, 65 °C, 10 s, slowly heating up to 97 °C, continuously measuring the fluorescence and cooling (40 °C, 30 s).Based on the tag position two different forward primers were used for envC-HA quantification.The reverse primer binds specifically in the HA-tag and thus can be used for SP-HA and RPep-HA (Table 3).Primer binding sites of the forward primers were selected so that amplicons suitable for qRT PCR are formed.

Reverse transcriptase activity test
The activity of viral RT in cell free SNs of PERV producer and infected cells was measured using the in-house HS-MN RT kit (Cavidi Ab, Sweden) in accordance to the manufacturer's instructions of protocol B. Samples were tested in triplicates (50 µL each) and mean value and standard deviation were determined.The standard dilution series covered RT activity values from 0.11 mU/ mL to 33.97 mU/mL.RT activity values below this were considered to be RT negative and defined the limit of detection.

Electron microscopy
EM of PERV-C-HA cells was performed according to standard procedures as described previously [21].Briefly, cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich, USA) in culture medium for 45 min at room temperature.Cells were washed two times with PBS.Subsequently, sample preparation was performed in the electron microscopy facility at Paul-Ehrlich Institut.Cells were scraped off the culture flask and gently mixed with 2% warm liquid agarose.After cooling and gelling, small agarose blocks containing the cells were cut and postfixed with 2% osmium tetroxide (Sigma-Aldrich, USA) in PBS and treated with 1% tannic acid (Carl Roth, Germany).Cells were gradually dehydrated in a series of ethanol baths and embedded in Epoxy resin (Sigma-Aldrich, USA).Polymerization lasted for 48 h at 60 °C, afterwards ultrathin sections were cut and stained with 2% uranyl acetate (Merck, Germany) for 15 min followed by 2% lead citrate (Serva, Germany) for 5 min.Sections were examined in a Jeol JEM 1400 Flash electron microscope using electron filtering (ESI) mode.

Integration control of PERV-C by PCR
Proviral PERV-C-HA was analyzed by PCR on purified gDNA from cells after superinfection.Target cell gDNA was purified using the QIAamp ® DNA Blood Mini Kit (Qiagen, Germany) as recommended by the manufacturer.PCR was performed with 100 ng gDNA with primer pairs EnvC for3 and rvEnvC-HA for PERV-C(5683)-SP-HA and EnvC for4 and rvEnvC-HA for PERV-C(5683)-RPep-HA.An EnvC specific PCR using the primers EnvC for3 and EnvC-R3_AWG was performed as control.gDNA of ST-IOWA cells served as negative control.Q5 DNA Polymerase (NEB, Germany) was used following manufacturers' instructions.

Structural prediction using AlphaFold2
Structural prediction of PERV-C(5683) Env as well as generated HA-tagged EnvCs was performed using Alpha-Fold2 via the ColabFold pipeline applying mostly default parameters [49,66].AlphaFold2 provides a computational approach that has been used to predict many protein structures with almost experimental accuracy [49].Resulting structure models gaining the highest model confidence for each protein were analyzed for structural changes using Chimera [67].pLDDT scores obtained after the AlphaFold prediction are shown in Additional file 3: Fig. S3.

Statistical analysis
Statistical significance of RNA samples was determined by Welch's t-test with 0.05 as the threshold.

Fig. 1
Fig. 1 Structure and design of PERV-C(5683)-HA constructs.The HA-tag was inserted into the env gene of PERV-C(5683) (KY352351.2) by site directed mutagenesis.A Regular structure of PERV-C(5683) env.PERV-C(5683) molecular clones carrying the HA-tag B C-terminal to the SP at amino acid (AA) 44-52 (SP-HA), C N-terminal to the VRA at AA 86-94 (HA-VRA) or D C-terminal to the RPep at AA 639-647 (RPep-HA).The integration sites of HA were chosen on the basis of functional studies[9,25,56,57] and secondary structure analyses using the open source tool PSIPRED[55]

Fig. 2 Fig. 2 (
Fig. 2 Quantification of vRNA and RT activity in ST-IOWA cells transfected with HA-tagged PERV-C(5683).Cells were monitored for 56 days by collecting cell culture SNs weekly.Expression of PERV-C(5683) A env and B pol vRNA for SP-HA and RPep-HA showed equal levels compared to the positive control PERV-C(5683) (ctr+).HA-VRA transfected cells revealed only basal expression comparable to the PERV-C negative ST-IOWA cells (ctr−).C Consistent to the presence of vRNA PERV-C(5683), SP-HA and RPep-HA became RT positive within two weeks after transfection.HA-VRA remained negative for RT activity.Significant means of < 0.05 according to Welch's t-test are indicated by (*).Analysis were performed in triplicates (See figure on next page.)

Fig. 3 Fig. 4
Fig. 3 Immunofluorescence and WB analysis of ST-IOWA cells transfected with PERV-C.A Immunofluorescence of SP-HA and RPep-HA transfected ST-IOWA cells using anti-HA-tag and anti-EnvC directed antibodies, 56 days p.t.. A1-4: ST-IOWA cells (ctr−), B1-4: PERV-C(5683) positive ST-IOWA cells (ctr+), C1-4: SP-HA and D1-4: RPep-HA transfected ST-IOWA cells.PERV-C Env proteins (red fluorescence) were detected for all cells except for the non-transfected control ST-IOWA.PERV-C Env-HA proteins were only observed for cells transfected with SP-HA or RPep-HA, as indicated by green fluorescence.B Detection of HA proteins by WB analysis in cell lysates of ST-IOWA cells transfected with SP-HA or RPep-HA harvested at day 56 p.t. (lane 1: ST-IOWA (ctr−), lane 2: SP-HA and lane 3: RPep-HA).Anti-beta-actin was used as loading control.PERV-C Env-HA precursor is expected at 72.1 kDa, its cleaved products SP-HA-SU at 52.3 kDa and TM-HA at 20.9 kDa.PERV-C Env-HA precursor proteins were detected for both, SP-HA and RPep-HA.SP-HA-SU shows only slight size variation compared to the precursor protein.For RPep-HA, both the precursor and the cleaved product were detected

Table 2 Fig. 5
Fig. 5 Infection of ST-IOWA cells with cell culture SN of HA-tagged PERV-C(5683).Cells were incubated for 24 h with SN and infection was monitored for 56 days on a weekly basis.Quantification of A envC and B pol vRNA showing increasing copy numbers from the first week on for PERV-C(5683) that was used as positive control as well as for SP-HA and RPep-HA.Non-infected ST-IOWA cells stayed on levels of basal expression.C Quantification of RT activity.RT activity could be detected from day 14 p.i. for all cell lines except for non-infected ST-IOWA cells that served as negative control (ctr−).D Immunohistochemistry of PERV-C-HA infected ST-IOWA cells at day 56 p.i. using PERV-C Env and HA-tag directed antibodies.A1-4 ST-IOWA (ctr−), B1-4 PERV-C(5683) infected ST-IOWA cells (ctr+), C1-4 SP-HA infected ST-IOWA cells, D1-4: RPep-HA infected ST-IOWA cells.In accordance with the transfection studies PERV-C Env-HA was only present in ST-IOWA cells infected with SP-HA or RPep-HA, indicated in green and yellow.Significant means of triplicates of < 0.05 according to Welch's t-test are indicated by (*)

Fig. 6
Fig. 6 Structural prediction of native PERV-C(5683) (KY352351.2) and HA-tagged PERV-C(5683) variants.Structures were predicted using AlphaFold2 [46] and compared using a MUSCLE alignment.PERV-C(5683) is colored in dark gray and PERV-C(5683)-HA variants are shown in light gray.Major sequence motifs are color-coded SP (green), VRA (light blue), VRB (dark blue), PRR (purple), and RPep (yellow).The HA-tag is colored in red.A Comparison and alignment of PERV-C(5683) and SP-HA.AI Whole proteins.AII Detailed view of predicted structural changes.Insertion of the HA-tag in SP-HA resulted in a shift of the PRR (left) and the formation of an additional alpha helix adjacent to SP (right).B Comparison and alignment of PERV-C(5683) and HA-VRA.(B I), whole proteins.(B II), detailed view of predicted structural changes.A twist in the PRR was observed in HA-VRA, resulting in a compressed loop compared with PERV-C(5683) (left).In addition, an intrinsically twisted loop was formed by the HA-tag (right).C Comparison and alignment of PERV-C(5683) and RPep.CI Whole proteins.CII Detailed view of predicted structural changes.RPep-HA shows a slightly shifted structure compared with PERV-C(5683).The main differences are in the SP, PRR (left) and RPep (right) and are indicated by arrow.pLDDT scores are shown in Additional file 3: Fig. S3

Fig. 7
Fig. 7 Quantification of vRNA and RT activity after challenging of ST-IOWA/PERV-C(5683) with PERV-C(5683)-HA viruses.Expression of PERV-C env vRNA was measured by qRT-PCR using A envC and B, C envC-HA specific primers.D Enzyme activity was monitored by RT assay.Native ST-IOWA cells were used as negative controls (ctr−), ST-IOWA cells infected with PERV-C(5683)-HA served as positive controls to confirm virus functionality.A Expression of envC vRNA was measured for all cells except for ST-IOWA (ctr−) starting from day 7 on.B, C envC-HA vRNA was detected for ST-IOWA cells infected with SP-HA or RPep-HA containing cell culture SNs but not for PERV-C(5683) positive cells challenged with PERV-C(5683)-HA.Significant means of < 0.05 according to Welch's t test are indicated by (*).Analysis were performed in triplicates

Fig. 8
Fig. 8 Immunofluorescence and WB analysis after superinfection.A Immunofluorescence of PERV proteins after superinfection of PERV-C(5683) positive ST-IOWA cells using antibodies directed against PERV-C Env and HA-tag.Protein expression was monitored at day 56 p.i.. A1-4: non-infected ST-IOWA (ctr−), B1-4: PERV-C(5683) positive ST-IOWA cells (ctr+), C1-4: ST-IOWA cells infected with SP-HA (ctr+) or D1-4: RPep-HA (ctr.+),E1-E4: ST-IOWA cells superinfected with SP-HA or F1-4: RPep-HA.PERV-C Env proteins were observed for all cells except for the (ctr−), while HA proteins only were present in primary infected control cells.B WB analysis of PERV-C proteins using antibodies directed against (a) the HA-tag or (b) PERV-C Env.WB was performed 56 days after superinfection of ST-IOWA-PERV-C(5683) cells with PERV-C(5683)-HA viruses.Native ST-IOWA cells were used as negative control (ctr−).ST-IOWA cells infected with virus present in SN that was also used for superinfection served as positive controls (ctr+).Lane 1: ST-IOWA (ctr−, lane 2: PERV-C(5683) positive ST-IOWA cells, lane 3-4: ST-IOWA cells infected with (3) SP-HA (ctr+) or (4) RPep-HA (ctr+), lane 5-6: PERV-C(5683) positive ST-IOWA cells superinfected with (5) SP-HA or (6) RPep-HA.A The EnvC-HA precursor (72.1 kDa) was detected for both SP-HA and RPep-HA.The corresponding cleaved product of SP-HA-SU is slightly below the precursor, thus indicated by a shadow.Since the HA-tag is located C-terminal in RPep-HA only the cleaved product TM-HA was detected.B For differentiation of EnvC and EnvC-HA, an anti-PERV-C Env(498) antiserum was used.PERV-C Env proteins were detected for all cells except for the negative control.C An anti-beta-actin WB was used as loading control days p.t.. Maximal RT activities were reached at day 42 with 78 mU/mL for SP-HA, 102 mU/mL

Table 3
Oligonucleotide primers used for qRT-PCR