Questions on unusual Mimivirus-like structures observed in human cells

Background: Mimiviruses or giant viruses that infect amoebas have the ability to retain the Gram stain, which is usually used to colour bacteria. There is some evidence suggesting that Mimiviruses can also infect human cells. Guided by these premises, we performed a routine Gram stain on a variety of human specimens to see if we could detect the same Gram positive blue granules that identify Mimiviruses in the amoebas. Methods: We analysed 24 different human specimens (liver, brain, kidney, lymph node and ovary) using Gram stain histochemistry, electron microscopy immunogold, high resolution mass spectrometry and protein identification. Results: We detected in the human cells Gram positive granules that were distinct from bacteria. The fine blue granules displayed the same pattern of the Gram positive granules that diagnose Mimiviruses in the cytoplasm of the amoebas. Electron microscopy confirmed the presence of human Mimiviruses-like structures and mass spectrometry identified histone H4 peptides, which had the same footprints as giant viruses. However, some differences were noted: the Mimivirus-like structures identified in the human cells were ubiquitous and manifested a distinct mammalian retroviral antigenicity. Conclusions: Our main hypotheses are that the structures could be either giant viruses having a retroviral antigenicity or ancestral cellular components having a viral origin. However, other possible alternatives have been proposed to explain the nature and function of the newly identified structures.


Introduction
There is evidence that terrestrial giant viruses can also infect mammals and a recent article published on Lancet Infectious Diseases describes the presence of giant viruses in human lymph nodes [1][2][3] . One of the chemical characteristics of giant viruses is their property to retain the Gram stain, which is usually used to colour bacteria 4,5 .
In fact, Mimiviruses (giant viruses) were initially mistaken for gram-positive bacteria infecting the cytoplasm of an amoeba, which was stuffed with blue Gram positive granules under an optical microscope. Only in 2003 did electron microscopy clarify that the fine blue granules present in the cytoplasm of the amoebas were actually giant viruses 6 .
Guided by the premise that giant viruses can also infect humans, we decided to perform a routine Gram stain on different human specimens to see if we could detect the same blue granules that were detected in the amoeba when Mimiviruses were first identified.
Here we demonstrate, with the use of electron microscopy, mass spectrometry and histochemistry, that human cells have anatomical areas that manifest some of the biochemical and morphological properties also found in giant viruses. These structures are ubiquitously present in a variety of human tissues, including non-pathological tissues. Possible alternative explanations of the findings are discussed.

Characteristics of human samples
• 3 liver specimens with haemochromatosis and non-alcoholic steatohepatitis • 1 liver specimen with cryptogenic cirrhosis (unexplained) • 7 liver specimens with chronic hepatitis B • 2 liver specimens with chronic hepatitis C • 3 liver specimens with non-specific minimal histological lesions • 2 liver specimens with no lesions • 2 liver specimens with primary biliary cirrhosis • 1 liver specimen from a patient with Crohn's disease Sample preparation for proteomics two dimensional gel electrophoresis Human samples were ground with liquid nitrogen. Six volume sample preparation buffer (9M urea, 2% ampholytes and 70 mM DTT) were added to the frozen powder, followed by three frozen/ thaw cycles (liquid nitrogen −196°C/30°C). After incubation for 30 min at room temperature and centrifugation for 45 min at 15000xg the supernatant was removed and frozen in new tubes at -80°C. FFPE slices were treated with 0.5 ml Heptan for 1h at room temperature. Subsequently, 25µl methanol were added and mixed for 25min. After centrifugation (5min, 13200xg) the pellet was air dried and 100µl lysis-buffer (250 mM Tris pH 9.5; 2% SDS) were added. The sample was boiled for 2h, centrifuged (30min, 13200xg, room temperature) and the supernatant was used for SDS-PAGE.
The 2DE gels used for comparison analysis were digitized at a resolution of 150 dpi using a PowerLook 2100XL scanner with transparency adapter.

Western blotting
For western blot applications, two identical gels were run. One 2DE gels was stained with FireSilver or Coomassie for preparative applications and the other gel was used for western blotting to detect the proteins by immunostaining. Blotting of 2DE gels was performed using an Immobilon-P membrane (PVDF; pore size 0.45 mm; Millipore) and a Trans-Blot SD Semi-Dry Transfer Cell (BioRad) at a constant current 5 V overnight at 4°C using a blotting buffer consisting of 25 mM Tris-HCl, 192 mM glycine, 0.1% SDS (pH 8.3) and 20% methanol. For immunodetection of proteins, membranes were washed in TBST (20 mM Tris-HCl [pH 7.5]; 154 mM NaCl, 0.1% Tween-20) and blocked in TBST containing 2% (w/v) BSA for 2 h. Membranes were incubated with the primary antibody (sc-65623; Santa Cruz Biotecnology; IgG 1 ) diluted 1:1000 for 2DE blot and 1:50 for 1D-blots in TBST containing 1% (w/v) BSA overnight and then incubated with antimouse IgG (Fc specific-peroxidase antibody produced in goat; A0168; Sigma; diluted to 1:2000 in TBST containing 1% (w/v) BSA) for 1 h at room temperature. Finally, the bound antibody was detected by incubating with Luminol for 1s-20min (Roth). The membrane was washed in TBST (5 times for 10 min) between all incubation steps.
Trypsin-in-gel-digestion/nanoLC-ESI-MS/MS Protein identification was performed using nano LC-ESI-MS/MS. The MS system consisted of an Agilent 1100 nanoLC system (Agilent), PicoTip electrospray emitter (New Objective) and an Orbitrap XL mass spectrometer (Thermo-Fisher). Protein spots from the membranes were in-gel digested by trypsin (Promega) (with and without citraconic anhydride treatment) and applied to nanoLC-ESI-MS/MS. Peptides were trapped and desalted on the enrichment column (Zorbax SB C18; 0.3x5 mm; Agilent) for five minutes using 2.5% acetonitrile/0.5% formic acid as eluent, then peptides were separated on a Zorbax 300 SB C18 column (75µmx150mm; Agilent) using an acetonitrile/0.1% formic acid gradient from 5 to 35% acetonitril within 40 minutes. MS/MS spectra were recorded data-dependently by the mass spectrometer, according to manufacturer's recommendations.

PEAKS and peptide identification
Immunoblot positive bands from frozen and FFPE tissues, were analyzed by mass spectrometry (nano LC-ESI-MS/MS), using a Thermo Orbitrap XL with CID fragmentation.
A database search was performed first against human proteins contained in UniProtKB/TrEMBL (http://www.ebi.ac.uk/uniprot) and virus proteins contained in UniProtKB/TrEMBL separately. After that, to reduce the risk of false positive results, the search was made against a combined human and viral database within a 1% false discovery rate. The search parameters were: 20 ppm precursor error tolerance, 0.6 Da fragment error tolerance, trypsin allowing non-specific cleavage at 1 end and a maximum of 3 missed cleaves, carbamidomethylation set as a fixed ptm, acetylation(k), oxidation (M), deamidation(NQ), formylation (K, Nterm), phosphorylation (STY) set as variable modifications.
The raw files were also processed through PEAKS Studio 8.0 (Bioinformatics Solutions Inc.) de novo and PEAKS DB modules. The parent mass error tolerance was set to 3 ppm, the fragment mass error tolerance was 0.6 Da. Carbamidomethylation of cysteine was set as a fixed modification and oxidation was set as a variable modification. The enzyme rules specified were trypsin, allowing non-specific cleavage at one end maximum and a maximum of three missed cleavages per peptide. The database searched was trEMBL (version is 2016_09). Only human and polydnaviridae proteins were searched, 1109386 protein sequences were searched along with a decoy database containing an equal number of proteins.

Gram positive staining
We Gram stained 21 different types of human liver specimens. We initially chose the liver, since this organ is the bio-chemical processing centre and the cross road of microbial invasions of the human body.
Gram positive blue granules were diffusely and ubiquitously expressed in all tested human liver samples, including unaffected liver samples with no histological lesions. These blue granules were absolutely distinct from common pigments, such as lipofuscin, and different from gram positive bacilli that were used as controls. The granules had a typically fine granular aspect, similarly to the one present in the amoebas infected by Mimiviruses, as reported by the French authors 6 . Figure 1 (premise picture) illustrates Gram positive granules that are Mimiviruses infecting amoebas. The permission to use this image was kindly provided by Prof Bernard La Scola.   ×80). After the Gram stain, human liver cells displayed fine blue granules that, for didactic reasons, are enclosed in the black circles, but they can be seen scattered in the parenchyma. Note the similarities between the amoebal Mimiviruses appearing as blue granules (small picture frame and Figure 1) with the human blue granules. In the human cells, the gram positive granules appear as fine granules and are distinct from bacteria and other pigment, like lipofucsin, that is also present (brown colour).
In the human liver cells, this fine blue granularity was detected in the cytoplasm and nuclei (Figure 2).
To further verify the ubiquitous presence of these typical blue granules in the human cells, we also Gram stained human brain, ovary, lymph node and kidney tissues. Granules could be detected in the kidney glomeruli, but not in the renal tubules (Figure 3). The brain was intensely stained, showing a diffuse granular pattern ( Figures 4A and B). The ovary did not display the gram positive granules ( Figure 5). In the lymph node, Gram positive granules were absent from the germinal centres and only appeared in the paracortex (Figures 6A and B).    Electron microscopy Subsequent electron microscopy (EM) analyses of the Gram positive human tissues confirmed the presence of cellular structures resembling Mimiviruses (Figure 7). This was exactly the same case of the French authors when they proved that the fine blue granules in the amoeba were actually Mimiviruses 6 . To enhance the resolution detection of the EM, we used a particular antigen retrieval solution with citraconic anhydride and heat 7,8 . EM analyses were conducted in two different international centres and 300 micrographs were scrutinized by operators who performed a blinded reading and were also blind to each other. The immunogold labelling assays revealed also a retroviral antigenicity associated to the structures when a mammalian anti-retroviral gag-p27 MoAb, recognizing common epitopes among several mammalian retroviruses, was tested.

Mass spectrometry
Mass spectrometry (nano LC-ESI-MS/MS) and protein identification using PEAKS 7.5 software revealed the presence within the structures of human proteins, including conventional human histone proteins that co-existed with a histone H4 peptide KTVTSM-DIVYALK. This manifested a distinct viral footprint of giant polydnaviruses that did not match any human sequence. In fact, the human and many other eukaryotes display in correspondence of their C-terminus histone-H4 tail a typical and extremely conserved sequence KTVTAMDVVYALK, with a I -> V replacement and human histone H4 variants that have never been described 9,10 .
To rule out false positive identifications when searching just with the virus database, we combined all identified proteins in the virus database and all identified proteins in the human database into one FASTA file (Supplemental File 1). The raw files were processed through PEAKS Studio 8.0, de novo and PEAKS DB modules.
When analysing the biological samples, the peptide KTVTSM-DIVYALK was identified confidently in two replicates at similar retention times: 23.05 minutes in replicate one and 23.37 minutes in replicate two.
To validate our results, a synthetic peptide with the same sequence as our candidate peptide KTVTSMDIVYALK was produced at the CRIBI peptide facility, University of Padua. A significant number of high intensity b and y ions matched the synthetic peptide spectrum. In particular, the b and y ion series from IVY (the part of the sequence that differs from the human protein), were prevalent in both spectra. We also performed a narrow scan in the mass range 730-740 m/z; MSMS for center mass 734.40 m/z (2 nd isotope of 733.9 m/z; z=2); MSMS for center mass 744.40 m/z (2 nd isotope of 734.9 m/z; z=2). The canonical human histone H4 and the IVY histone H4 variant were both present at m/z= 734.907; z=2. A summary of the proteomics assays are reported in Figure 8- Figure 12 and in Supplemental File 2.
Three dimensional (3D) protein models of the canonical human histone H4 protein and the histone H4 isoform having the viral footprint were generated by using the Swiss Model (https://swissmodel.expasy.org/).

Discussion
Although there are morphological and biochemical properties similar to giant viruses, the newly identified structures are possibly beyond the concept of typical viruses. The structures are ubiquitous in human tissues and are not associated to a specific medical disease. We are aware that being ubiquitous does not necessarily mean that these structures are not viruses and not being infectious does not imply that they are not viruses, since viruses can be also ubiquitous and not pathogenic 11-13 . However, the type of the histological pattern and the mass spectrometry identification do not completely rule out that these structures could be human cellular components having a viral footprint [14][15][16] . Like mitochondria that were originally bacteria cells and still retain the bacterial features 17-19 , the human Mimivirus-like structures manifest an  Section 1: The identified peptides are the canonical human histone-4 isoforms. Section 2: The histone H4 peptide KTVSMDIVYALK, indicated by the green arrow, has a unique footprint not found in any human or other eukaryotes proteins. This peptide displays the same unique sequence found in the C-terminus of the histone H4 of giant polydnaviruses (purple colour in the alligmnements between eukaryotes and polydnaviruses histone H4 sequences).
ancestral origin. Some of the histone variants detected within the human structures have the same universal motifs associated to the same function that are also used by giant polydnaviruses to manipulate their host transcription. The IVY histone pattern, that is present in these structures, tells the cells that some genes should be "off".
The basis for this assertion corresponds to the findings of an identical IVY pattern in giant viruses that represses host gene transcription 20-22 . In addition, the three-dimensional analysis of the histone H4 that displays the IVY sequence shows a closed conformation that might prevent gene transcription (Figure 13). It would be interesting to trace if an evolutionary link may exist between these human cellular structures, giant viruses or archaea. The recent finding that giant viruses can integrate into modern eukaryotic genomes have motivated the fascinating and highly provocative idea that giant viruses, along with archaea and bacteria, contributed significantly to the evolution of the first eukaryotes 23-26 .

Conclusions
In conclusion, did we find ubiquitous giant viruses suppressing human responses or human structures with "something that was originally giant" and are not viruses any longer? The ancestral nonhuman nature of these structures is supported by the IVY histone pattern identified with mass spectrometry and by their capability to retain the Gram stain, which colour peptidoglycans. However, there are other alternative explanations for the structures that need to be considered as well. For example, the documented mammalian retroviral antigenicity does not entirely exclude the possibility that these structures could represent particles formed by the concurrent activity of retro-transposons.
By the virtue of development of the science of microscopy, the ultrastructure of the cell apparatus has been established by the 1960. Since then, new structures have been sporadically reported. The main challenge when uncovering cellular components is proteomics, which can be technically much more complex than transcriptomics, and electron microscopy is perceived by some scientists as an old fashioned technique prone to artefacts. However, it is worth mentioning that the Golgi apparatus was discovered with the use of a rudimental microscope in 1898 and many scientists did not believe that the Golgi apparatus was real and instead argued that the apparent body was a visual distortion caused by staining 27-30 . It took almost a century to fully understand the function of the Golgi apparatus. The aim of this paper is to merely report what we have found inside the human cells and offer some hypotheses. Only time and additional experiments will clarify if the identified structures are giant viruses having a retroviral antigenicity or cellular components having a viral ancestry or human retrotransposon-like elements.

Data availability
All the histological samples, slides, EM grids are available to be examined; please contact the corresponding author.
Author contributions EAL: conceived and led the research, protocols strategies, data analyses, manuscript; DM: mass spectrometry analyses, use of the PEAKS software; CF: Electron Microscopy Analyses; PG: Histochemistry, pathology reading, patient analyses and materials collection.

Competing interests
No competing interests were disclosed.