Spuma or foamy viruses (FVs), which constitute several genera in the retrovirus subfamily Spumaretrovirinae
], display a replication strategy with features common to both other retroviruses (Orthoretrovirinae
) and hepadnaviruses (reviewed in [2
]). FVs are unique amongst retroviruses, as the initiation of reverse transcription (RTr) of the packaged viral genomic RNA (vgRNA) occurs in a significant fraction of virions (5–10%) during viral assembly [4
]. Thereby, unlike to orthoretroviruses, both vgRNA and/or viral genomic DNA (vgDNA) containing virions are found in the supernatant of FV infected cells. It is generally accepted that the vgDNA containing virions contribute to the majority of new productive infection events during spreading of FVs in cultures, at least in vitro [5
]. However, a low level of reverse transcription, probably derived from vgRNA containing virions, has been observed during uptake of FVs at very low multiplicities of infection (MOI) [4
FVs are naturally endemic to most non-human primates (NHPs), including New and Old World monkeys and apes, cats, cows, horses, tree shrews, sea lions, and bats (reviewed in [2
]). In addition, endogenized copies of FV genomes were identified in sloths, the aye-aye, the Cap golden mole [10
], cod [12
], platyfish [12
], zebra fish, and the coelacanth. Nowadays, humans are not considered as a natural host, but frequent zoonotic transmissions of NHP simian FVs (SFVs), but not feline FV (FFV) or bovine FV (BFV), have been observed in workers occupationally exposed to NHPs—bush meat hunters in central Africa, and in various contexts of human–NHP interspecies contact in South and Southeast Asia. Cases of spread from human to human have not been reported. The best-studied and characterized isolate to date is the so-called prototype FV (PFV; formerly known as human FV, HFV), which was originally isolated from an African patient who presumably was infected zoonotically by a chimpanzee FV [13
Another characteristic of FVs is their extremely broad tropism. In vitro, only very few species and cell types are known to be non-permissive to FVs or FV Env-mediated entry [16
]. FV infection in vitro is highly cytopathic to most cell types, except cell lines or primary cells of myeloid or lymphoid origin, which can become chronically infected [17
]. The cellular targets of FVs in vivo remain poorly characterized. In infected monkeys, the viral genome is detectable in many tissues but appears to be largely in a latent state, as viral replication is reported to be mainly restricted to the superficial epithelial layer of the oral mucosa [19
]. This explains the major transmission mode between monkeys and zoonosis to humans through bites. In the blood, proviral DNA is detected in CD8+
- and CD4+
T-cells (memory and naïve) as well as B-cells, and to a lower extent also in monocytes and NK-cells [20
]. Other types of immune cells that encounter FVs during the course of an infection in vivo have not been characterized.
FVs have co-evolved with their hosts for at least 60 million years and are considered to be non-pathogenic in natural hosts and zoonotically infected humans. The immune system seems to control FV infection very efficiently in natural hosts and zoonotically infected humans, as replication and viral load of FV stays low [20
]. However, the mechanisms involved are poorly understood (reviewed in [8
]). In particular, the interaction of FV with the innate immune system remains to be fully clarified. FVs are known to respond in vitro in a cell type-dependent manner towards IFNs [23
]. Furthermore, treatment of cells of different origin with type-I or II IFNs, impairs spreading of FVs or inhibits early steps in FV replication [24
]. In line with this observation, restriction of FVs by several IFN-induced cellular proteins has been reported, although their relevance for FV replication in vivo has not been tested so far [29
]. Although FV replication in vitro is impaired by addition of exogenous IFNs, infection of a variety of tissues does not seem to mount an innate immune response [28
]. Only recently, stimulation of IFN secretion by human hematopoietic cells, in particular, plasmacytoid dendritic cells (pDCs), upon incubation with SFV virions or SFV infected cells, was demonstrated [23
]. pDCs, as the main producer of type-I IFN, detect FV RNA by the TLR7-mediated pathway. However, the contribution of myeloid cells, besides pDCs, to innate sensing and IFN induction has as of yet not been investigated. Above all, conventional dendritic cells (DCs) play a critical role in detecting retroviruses, as shown for the lentivirus HIV-1 [33
], and promote, thereby, the activation of adaptive immunity [34
]. For HIV-1 and other lentiviruses, it has been demonstrated that the reverse transcribed DNA products generated upon viral entry in DCs mediate the activation of transcription factors, such as IRF3, resulting in ISG expression and IFN synthesis [33
]. Thereby, the viral reverse transcribed components are sensed by the DNA sensor cyclic GAMP synthase (cGAS) [36
] together with polyglutamine binding protein 1 (PQBP1) [37
]. Interestingly, and in contrast to lentiviruses, FV infected cells release both vgRNA and vgDNA containing virions, and harbor RTr products late during the assembly steps. Therefore, exposure of DCs and other myeloid cells to FVs may result in interactions with the innate immune system different to those of other retroviruses. For instance, they could include pathways encountered by hepatitis B virus (HBV), as HBVs, like FVs, reverse transcribe their genome during particle morphogenesis (reviewed in [38
The aim of this study was to investigate whether the innate immune system of cells of the human myeloid lineage is capable of sensing FVs. If so, the viral pathogen-associated molecular patterns (PAMPs), the pattern recognition receptors (PRRs), and sensing pathways involved were to be characterized. We found that the innate immune system responds to sensing of RTr products of full-length PFV genomes but not of minimal vector genomes in the cytoplasm of human myeloid cells with an efficient interferon-stimulated gene (ISG) induction within hours of virus exposure. Sensing of PFV RTr products was dependent on cellular cGAS and STING expression and largely insensitive to reverse transcriptase inhibition during viral entry, suggesting that the already viral DNA (vDNA) containing PFV particles are the main stimulator.
2. Materials and Methods
2.1. Cells and Culture Conditions
The human embryonic kidney cell line 293T (ATCC CRL-1573) [39
] and a proteoglycan-deficient variant 293T-25A (described elsewhere), the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [40
], and the clonal variant HT1080 PLNE containing a PFV LTR driven EGFP
reporter gene expression cassette [41
], were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v
) heat-inactivated fetal calf serum and antibiotics. The human monocyte cell line THP-1 (ATCC TIB-202) and knockout (KO) variants ∆IFNAR1, ∆SAMHD1 [42
], ∆cGAS [43
], ∆MAVS [43
], ∆MyD88, and ∆STING [43
] were cultivated in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) supplemented with 10% (v
) heat-inactivated fetal calf serum, antibiotics, 2.5 g/L glucose, 10 mM Hepes, and 10 mM sodium pyruvate. THP-1 cells were differentiated into macrophage-like cells by the addition of phorbol-12-myristyl-13-acetate (PMA) at 30 to 50 ng/mL final concentrations for 48 h prior to exposure of viral supernatants.
Human buffy coats, of anonymous blood donors, were obtained from German Red Cross Blood Donor Service Baden–Württemberg Hessen. Primary human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using Ficoll density gradient and subsequent isolation of CD14+
]. Briefly, PBMCs were separated by density gradient centrifugation (30 min, 980× g
, RT), using Ficoll (Histopaque 1077 Sigma-Aldrich Biochemie GmbH, Hamburg, Germany). The mononuclear layer of the interphase was recovered and washed twice with PBS. PBMCs were subsequently incubated with 0.86% (w
) ammonium chloride for erythrocyte lysis (37 °C, 10 min). PBMCs were washed twice and filtered (0.7 µm). CD14+
monocytes were positively selected using CD14-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol, and separated from unlabeled cells using an AutoMACS device (Miltenyi Biotec). Subsequently, cells were differentiated into monocyte derived dendritic cells (MDDC) or monocyte derived macrophages (MDMs) by cultivating for 5 days in RPMI-1640 medium supplemented with 2 mM l
-Glutamine; 10% (v
) FCS; 1% (v
) HEPES, 1 mM sodium pyruvate; 280 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine®
Sargramostim, Genzyme, Boston, MA, USA); 800 U/mL IL-4 (PeproTech GmbH, Hamburg, Germany) for MDDCs and 560 IU/mL GM-CSF only for type 1 proinflammatory MDMs. The same amounts of medium and cytokines were added on day 3. On day 5 of differentiation, MDMs were detached by a short incubation with PBS-EDTA and MDDCs by gentle resuspension in PBS; they were counted and plated for cell stimulation experiments and simultaneously checked for MDDC and MDM surface markers and their differentiation status.
2.2. Recombinant Plasmid DNAs
A four-component PFV vector system, consisting of the expression-optimized packaging constructs pcoPG4 (PFV Gag), pcoPE (PFV Env), and pcoPP (Pol), and an enhanced green fluorescent protein (EGFP)-expressing PFV transfer vector pMD9 or puc2MD9, has been described previously [16
The CMV-driven proviral expression vector pczHSRV2 (wt) and its variants pczHSRV2 M69 (iRT), expressing a Pol protein with enzymatically inactive RT domain (YVDD312–315
GAAA mutation), pczHSRV2 M73 (iIN), with enzymatically inactive IN domain (D936
A mutation), and pczHSRV2 EM271 (∆Env), with inactivated Env translation start (M1T, ATG to ACG, M5T, ATG to ACG; M16T, ATG to ACG mutation) were described previously [5
]. For this study the variants pczHSRV2 EM284 (∆Gag), with inactivated Gag translation start (M1L, ATG to CTG; S3Stop, TCA to TAA mutation); pczHSRV2 EM270 (∆Pol), with inactivated Pol translation start (M1L, ATG to CTG mutation); pczHSRV2 EM273 (∆GPE), with simultaneously inactivated Gag, Pol, and Env translation starts; pczHSRV2 EM020 (iFuse), with inactivated Env SU/TM furin cleavage site (R571T mutation); and pczHSRV2 EM010, with inactivated Tas translation start (M1L, ATG to TTG mutation), were generated. All constructs were verified by sequencing analysis. Primer sequences and additional details are available upon request.
For VLP-Vpx production the lentiviral vector pSIV3+, derived from SIVmac251 was used, as previously described [49
]. For single-round HIV-1 reporter virus production the plasmid pBR-NL43-Env−
] was used. In both cases the envelope vector pCMV-VSVg was used for pseudotyping.
2.3. Transfection, Virus Production, and Titration
Cell culture supernatants containing recombinant PFV particles and respective mock controls were generated by transfection of the corresponding virus encoding or mock control plasmids (mock A: pUC19; mock B: pczHSRV2 EM273 (∆GPE)) into 293T cells using polyethyleneimine (PEI) as described previously [41
], or 293T-25A cells using calcium phosphate (described elsewhere). Cell-free supernatants were generated by passing through a 0.45 µm filter and were either stored in aliquots at −80 °C when used for stimulation experiments or processed further for additional analysis. Viral titers of recombinant, EGFP-expressing PFV vector particles (PFV-SRVs) by fluorescence marker-gene transfer assay on HT1080 cells were determined as described previously [51
]. Virus particles generated by use of proviral expression plasmids (PFV-RCPs) were titrated on HT1080 PLNE cells harboring a Tas-inducible nuclear EGFP
ORF in their genome as described previously [41
VLP-Vpx and HIV-1 GFP reporter viruses were produced as previously described [52
]. Briefly, 2 × 107
HEK293T/17 cells per T175 flask were seeded. The next day, 15.2 µg pSIV3+ and 2.3 µg pCMV-VSVg for VLP-Vpx production and 11.6 µg of pBR-NL43-Env−
and 5.9 µg pCMV-VSVg, for HIV-1 reporter virus production, per flask, were transfected using 18 mM PEI (Sigma-Aldrich). Medium was changed approximately 16 h later and viral supernatants were harvested 48 and 72 h post-transfection. Supernatants were centrifuged (10 min at 4 °C; 1500 rpm), filtered (0.45 µm), and DNaseI digested (1 U/mL) for one hour. Viral supernatants were purified by ultracentrifugation through 20% (w
) sucrose (2 h, at 4 °C; 25,000 rpm); virus pellets from day one and two were resuspended in PBS, pooled, aliquoted and stored at −80 °C. HIV-1 reporter viruses were titrated via serial dilutions on TZM-bl reporter cells using the beta-galactosidase colorimetric assay. VLP-Vpx were titrated according to their ability to target the restriction factor SAMHD1 for degradation, using Western blot.
2.4. Myeloid Cell Stimulation and qPCR Analysis of ISG Induction
For stimulation experiments, THP-1 cells were plated at 2 × 106 cells/well (3 × 104 cells/well) in a total volume of 2 mL (100 µL) in 6-well (96-well) plates and PMA was added to 30 ng/mL (50 ng/mL) final concentration. Forty-eight hours later the medium was replaced by the respective virus supernatant, mock control supernatants (mock A: pUC19, mock B: PFV-RCP ∆GPE mutant) or medium (medium) as indicated. At different time points post virus exposure, as indicated, viral supernatants were aspirated and cells were snap frozen at −80 °C and stored until subsequent nucleic acid extraction. MDDCs and MDMs were plated at 3 × 104 cells/well in the differentiation medium without cytokines, 24 h prior to infection. Virus supernatants, either PFV or a VSV-G pseudotyped full-length HIV-1 GFP reporter virus, with or without the addition of VLP-Vpx were added. AZT (Sigma) was added during infection in the indicated experiment at a final concentration of 100 µM. MDDC and MDM infections were conducted by spinoculation at 1200 rpm, 32 °C for 1.5 h. Viral supernatants were replaced by fresh medium and cells were cultivated at 37 °C, 5% CO2. At different time points post infection, cells were either lysed for RNA extraction and subsequent RT-qPCR or stained for FACS analysis.
2.5. Quantitative PCR Analysis
Cellular nucleic acids from cultures in 6-well plates were extracted using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. qPCR analysis of cellular mRNA expression using Maxima Probe qPCR Master Mix including ROX dye (ThermoFisher Scientific, Dreieich, Germany), a StepOnePlus (Applied Biosystems, Foster City, CA, USA) quantitative PCR machine, and plasmid standard curves was performed as previously described [48
]. Primers, Taqman probes, and cycling conditions are summarized in Table A1
. Cellular nucleic acids from cultures in 96-well plates were extracted using NucleoSpin®
RNA Plus Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Relative expression levels of ISG54
were determined using QuantiTect SYBR Green RT-qPCR Kit (QIAGEN) with the respective specific primers on a LightCycler®
480 Instrument (Roche, Basel, Switzerland). Relative mRNA expression levels were normalized to the housekeeping gene RPL13A
and analyzed using the 2^(−∆∆CT) method, finally depicted as fold inductions over mock A, mock B, or medium, as indicated. Primers and cycling conditions are summarized in Table A2
. Primer efficiencies have been tested before in 10-fold serial dilutions and were calculated to have >90% efficiency.
2.6. Flow Cytometry Analysis
Purity of MDMs and MDDCs was assessed via flow cytometry analysis. Triple stainings, of 1 × 105
cells with CD14-Pacific blue (BioLegend, San Diego, CA, USA), CD163-PE (BD), CD206-APC (BD) and CD1a-PE (BioLegend), and CD11c-Vio Blue (BioLegend) and CD16-APC (BioLegend) were performed with the matching IgG controls, listed in Table A3
. In order to determine CD86 activation, marker expression upon infection with different PFV mutants, 24 h post infection, 6 × 104
cells were stained with CD86-PE (Biolegend) or the corresponding isotype control. Briefly, after 5 days of differentiation, MDMs were detached by a short incubation with PBS-EDTA, MDDCs by gentle resuspension in PBS. Cells were washed twice with FACS staining buffer (PBS containing 10% (v
) FCS), FC-Block (1:10, BD) was added to prevent unspecific binding via Fc-receptors (10 min, RT), cells were resuspended in the specific antibody dilutions or corresponding isotype-controls, and they were stained for 20 min on ice. Subsequently, cells were washed twice with FACS staining buffer, fixed with ice-cold 2% (w
) Paraformaldehyde, washed twice, and analyzed via flow cytometry using MACSQuant Analyzer 10 (Miltenyi) and FCS Express software (De Novo Software, Glendale, CA, USA). For MDM and MDDC purity, percentages of positive cells of the specific markers are depicted in Figure S1
. For CD86 expression of infected MDDCs, mean fluorescent intensities (MFIs) normalized to the IgG controls of each condition are depicted as relative MFIs normalized to the wt treatment. Infection levels by HIV-1 GFP with and without AZT were assessed by determining the percentage of GFP-positive cells (Figure S2
). The cut-off was set to 0.1% with the IgG controls or the non-infected controls.
2.7. Analysis of PFV Particle Protein and Nucleic Acid Composition
PFV particles were concentrated from cell-free supernatants by centrifugation at 4 °C and 25,000 rpm for 3 h in a SW32Ti rotor (Beckman Coulter GmbH, Krefeld, Germany) through a 20% (w
) sucrose cushion. The particulate material was resuspended in phosphate-buffered saline (PBS) and used immediately for further analysis or stored at −80 °C. Western blot analysis was performed after protein sample buffer addition as described before using PFV Gag and PFV Env leader peptide (LP) specific antisera [48
]. DNase digestion of viral particles, extraction of particle-associated nucleic acids, and analysis of nucleic acid composition by qPCR was done using the primer–probe sets listed in Table A1
, as described before [46
2.8. Analysis of Cellular Protein Expression
For immunoblot analysis of IRF3 phosphorylation and SAMHD1 degradation, 5 × 105 MDDCs/12 wells were seeded and exposed to either PFV-RCP or VLP-Vpx. Six or twenty-four hours post-exposure, cells were harvested by resuspension in ice cold PBS, centrifuged (300× g, 6 min, 4 °C), and lysed in 25 µL RIPA-lysis buffer (100 mM NaCl; 10 mM EDTA (pH 7.5), 20 mM Tris (pH 7.5); 1% (v/v) Triton X-100; 1% (w/v) sodium deoxycholate) containing protease and phosphatase inhibitor cocktails (Complete Protease Inhibitor Cocktail; PhosSTOP Phosphatase Inhibitor Cocktail, Roche) for 45 min on ice. Lysates were centrifuged (17,000× g for 15 min at 4 °C), and protein concentration was determined based on the Bradford assay using the Bio-Rad Protein Assay Dye Reagent Concentrate. Samples containing 20 µg protein were prepared with NuPAGE LDS sample buffer (4×) and NuPAGE Sample Reducing Agent (10×), to a final 1× concentration and denatured at 70 °C for 10 min. Proteins were separated on precasted NuPAGE™ 4–12% Bis-Tris gradient gels (Invitrogen). The gel was run in 1× MOPS buffer (1 M MOPS, 1 M Tris, 69.3 mM SDS, 20.5 mM EDTA Titriplex II) supplemented with 200 μL NuPage Antioxidant 10× (inner chamber) at 200 V for 1 h 10 min. Proteins were transferred to a Hybond P 0.45 PVDF membrane (GE Healthcare, Chicago, IL, USA) using the XCell IITM blotting system with 1× NuPAGE transfer buffer (Invitrogen) at 35 V for 1 h 40 min. Membranes were blocked in 5% (w/v) BSA (Carl Roth) in 0.01% (v/v) Tris-buffered saline with Tween 20 (TBST) for 2 h at 4 °C with subsequent incubation in primary antibody dilutions at 4 °C overnight. Horseradish peroxidase (HRP)-linked goat anti-rabbit or horse anti-mouse IgG (heavy and light chain) secondary antibodies (Cell signaling, Danvers, MA, USA) were applied for 2 h at 4 °C. For detection Pierce® ECL Western Blotting Substrate (ThermoFisher Scientific) or ECL Prime (GE Healthcare) were used and the emitted chemiluminescence was detected at different exposure times on autoradiography films (Amersham Hyperfilm ECL, GE Healthcare). The following primary antibodies were used and applied at 4 °C, overnight: Anti-Phospho-IRF-3 (Ser396) (Cell Signaling, number 4947); Anti-IRF3 (Epitomics, number 2241-1); Anti-GAPDH (Cell Signaling, number 2118); and Anti-SAMHD1 (Proteintech; number 12586-1-AP). In order to remove phospho-IRF3 antibody, probed membrane was incubated in stripping buffer (2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 100 mM β-mercaptoethanol), rotating for 45 min at 65 °C.
All the statistical analyses were performed using GraphPad Prism 8. The numbers of experimental replicates and information on the statistical methods used for determination of two-tailed p-values are described in the individual figure legends. Symbols represent: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant (p ≥ 0.05).
FV infections of natural hosts and zoonotic transmission to humans appear to be efficiently controlled by the immune system. We have only limited knowledge on the immunological mechanisms involved in this process.
Here, we examined the interaction of PFV with the innate immune system in immune cells of the myeloid lineage. Using an in vitro model cell line, THP-1, and primary human MDDC and MDM cultures, we observed an efficient stimulation of the innate immune system, determined as an IRF3-dependent stimulation of ISG expression and IRF3 phosphorylation, by replication-competent PFV generated from proviral expression constructs (PFV-RCP).
Furthermore, PFV innate sensing was neither dependent on viral transactivator Tas-mediated de novo viral transcription nor on viral integrase enzymatic activity. This indicates that productive infections, late steps of the viral replication cycle and virus spreading are not a prerequisite for PFV innate sensing in myeloid cells.
By use of various PFV-RCP mutants we demonstrated that PFV sensing occurs predominantly in the cytoplasm of myeloid cells as fusion-defective PFV-RCPs failed to stimulate ISG expression.
In contrast, efficient ISG induction required an enzymatically active reverse-transcriptase, indicating that vDNA or RTr products generated during reverse transcription are the major PFV PAMPs sensed by the innate immune system. Since FV RTr is observed both late in the replication cycle after capsid assembly and early during host cell entry [4
], we investigated whether vDNA and RTr products already being present in PFV particles or those newly generated during uptake are the major PAMPs. Inhibition of RTr during entry by RT inhibitor led only to a minor reduction in PFV-mediated ISG induction, whereas that of HIV-1 was strongly reduced. This indicates that the vDNA and RTr products already present in PFV particles are sufficient for efficient induction of an innate immune response. However, since we are unable to prevent RTr during PFV assembly and at the same time allow subsequent RTr to take place during virus entry, as AZT incorporation leads to dead-end products, we cannot formally exclude that the latter may contribute to a certain extent to the innate immune response.
In line with the ISG induction potentials of the various PFV-RCP mutants, we observed that inactivation of essential key molecules of cellular DNA-sensing pathways, cGAS and STING, in THP-1 cells, abolished PFV-RCP-mediated innate immune stimulation. In accordance with vDNA and RTr products already present in PFV particles before host cell entry, representing the main PFV PAMPs, inactivation of cellular SAMHD1, either by gene KO in THP-1 cells or VLP-Vpx co-delivery in MDDCs, had only minor negative effects on PFV-RCP mediated ISG induction. This is also in agreement with previous reports of SAMHD1, unlike for lentiviruses, not being a restriction factor for PFV [59
pDCs were shown by Rua and colleagues to mount an innate immune response as a consequence of TLR7-mediated sensing of PFV RNA [23
]. Our results suggest that in myeloid cells, the contribution of vRNA sensing to PFV-mediated innate immune stimulation appears to be negligible. This is underlined by clearly detectable, although slightly reduced ISG induction in THP-1 KO cells having key molecules of cellular RNA-sensing pathway, MAVS or MyD88, inactivated. Furthermore, PFV-mediated ISG induction was almost completely abolished when myeloid cells were incubated with PFV-RCP with enzymatically inactive RT, which did not contain vDNA but harbored similar levels of vRNA as wild type virus.
The most striking finding of this study was the requirement of vDNA and/or RTr products to be derived from full-length vRNA genomes (PFV-RCP) instead of RNA genomes with minimal cis-acting viral sequences of PFV single-round vectors (PFV-SRV) for efficient ISG induction in myeloid cells. Interestingly, in contrast to the minimal genome of single-round vectors (PFV-SRV wt), which had a strongly impaired ISG induction capacity, the RTr of single-round vectors encapsidating a full-length genome containing point mutations (PFV-RCP ∆GPE + G/P/E) led to an ISG induction profile similar to replication-competent, wild type PFV particles (PFV-RCP wt). Our results obtained with various kinds of PFV-SRVs and PFV-RCP mutants can rule out differences in the replication capacity, the encoding of structural proteins, and the particle-associated RT levels or vDNA copy numbers as causative for this difference in innate stimulatory capacity. This underscores that most likely the encapsidated and reverse transcribed full-length genome represents the immunostimulatory component. Currently we can envision several potential underlying mechanisms.
A very attractive but perhaps also the most unlikely explanation might be the presence of immunostimulatory determinants in full-length PFV genomes that are absent in minimal PFV vector genomes. A specific viral stimulatory sequence element (vSSE), and/or secondary structure thereof, present only in RTr products of full-length PFV genomes, could potentially be sensed.
Alternatively, the size difference between full-length PFV proviral (11,024 nt) and minimal SRV genomes (4348 nt), and not, or not only, a specific vSSE absent in the latter, may be responsible for or contribute to their differential ISG induction potential. This would fit to reports of cGAS activation based on DNA length and based on long DNA pre-structured by host proteins to strongly stimulate DNA sensing [60
]. HIV-1 minus strand strong stop DNA ((-)sssDNA) was reported to contain short stem-loop structures with flanking unpaired guanosines highly stimulatory for cGAS activity. Notably, full-length PFV-RCP and minimal PFV-SRV genomes are identical up to the translation start of the gag
ORF, thereby leading to identical PFV (-)sssDNA’s (Figure A1
). Therefore, even if PFV (-)sssDNA harbors cGAS stimulatory structures analogous to HIV-1 (-)sssDNA, it cannot be the cause for the differential ISG stimulatory capacity of RTr products of full-length PFV-RCP compared to minimal PFV-SRV genomes.
Finally, we cannot rule out that currently unknown differences in stability, structural integrity, or uncoating of the infecting viral cores containing full-length wild type or point mutation-containing genomes in comparison to minimal vector genomes are causing the difference in the innate response.
Further studies, including a detailed bioinformatic analysis of secondary structure prediction of PFV RTr products and their experimental verification, are required to provide experimental evidence for any of the proposed mechanisms, and combinations thereof, or they may reveal a currently unknown way of innate sensing of FV vDNA or RTr products and may identify additional cellular factors involved in this process.