Electron micrograph and immunogenicity profile of purified plant HPV16 L1 VLPs.

<p>Purified (V3) sample <b>(A)</b> and HPV16 VLPs derived from insect cells (<b>A1</b>) were absorbed on carbon coated grid and negatively stained with 2% phospho-tungstic acid (pH 6.8). Magnification was 46,000x; <b>(B)</b> The ELISA profile of hyperimmune sera collected from plant HPV16 L1 VLPs immunized mice. First two bars show the generated immune response against the sera of unimmunized mice (UM) act as a control and immunized mice with plant purified HPV16 VLPs, respectively when plant VLPs were loaded as antigens onto ELISA plate. Third and fourth bar represents the insect cell VLPs response, coated as antigens, against the sera collected from the above unimmunized (UM) and immunized mice (IM) with plant VLPs, respectively. Student’s t-test was done to compare differences between unimmunized (UM) and immunized mice (IM) from VLPS of plant extract and insect cells.</p

Diagrammatic presentation of constructed protein expression vectors for transformation of <i>Agrobacterium</i> and plants.

<p><b>(A)</b> Pictorial representation of constructed vectors. Restriction digestion profile of cloned plasmids DNAs (V1, V2, and V3) from <i>E</i>. <i>coli</i> (Lane 1–3) <b>(B)</b> and PCR amplifications of transformed <i>E</i>. <i>coli</i> (Lane 1–3) and <i>Agrobacterial</i> cells (Lanes 4–6), <b>(C),</b> respectively Target DNAs (1.5 and 1.7 Kbs) were found on 1% agarose gel when compared with DNA molecular weight marker (Lane M, 1 kb DNA ladder, Invitrogen).</p

Purification profile of V3 infiltrated tobacco plants.

<p><b>(A)</b> Immunoblot showing the target L1 protein collected from CsCl density gradients top (TB) and lower bands (LB) from V3 infiltrated plants. <b>(B)</b> The size exclusion chromatogram of CsCl collected and dialyzed V3 sample on 16/600 superdex-200 prep grade column using FPLC system. The molecular standards represented on the chromatogram are BD; bluedex (2000 kDa), C; conalbumin (75 kDa), O; ovalbumin (43 kDa). <b>(C)</b> SDS-PAGE and immunoblot showing the L1 band of dissociated chromatographic purified VLPs. The VLPs from insect cells were used as loading positive control and arrows in immunoblots show HPV L1 band at 56 kDa; 16E was used as HPV16 L1 sequential epitopes detecting monoclonal antibody. <b>(D)</b> ELISA profile for characterization of plant purified HPV16 VLPs using monoclonal (16A, C, and E) and polyclonal (R intact) in-house antibodies. 16A and C detect conformational epitopes, whereas 16E and R intact detect both conformational, as well as sequential epitopes. (<b>E)</b> Validation of purified VLPs by immunoblot using the same sequential and conformational antibodies as described in D. One way analysis of variance (ANOVA) was done to appreciate the dose response and a value *<i>p</i> < 0.05 was considered significantly different.</p

The detection profile and quantification analysis of L1 protein extracted from transformed tobacco plants.

<p>The immunoblots represent the HPV16 L1 detection profile of different vector-infiltrated plants extracted with <b>(A)</b> HEPES buffer, <b>(B)</b> using different physiological conditions with pH 2–8 in the presence (WS) or absence of salt (NS), <b>(C)</b> extraction of HPV16 L1 protein with PBS buffer at 5–9 days post infiltration (DPI) from plant leaves infiltrated with construct (TPL1F; V3). Antibodies, MoAb-16E and PoAb- disCPV2 were used for immunoblots. The positive control (PC) was disrupted VLPs from insect cells and arrows in immunoblots show HPV L1 band at 56 kDa. <b>(D)</b> Bar diagram of ELISA for VLPs extracted from crude plant extract (V3) at 5–9 DPIs. MoAbs-16 A, C and E and PoAb-R intact were used to detect specific sequential and conformational epitopes of HPV16 L1 and VLPs from insect cells were used as a positive control (PC). Student’s t-test was done to compare VLPs from plant extract at 5–9 DPIs with VLPs from insect cells. *<i>p</i> < 0.05; **<i>p</i> <0.01 and ***<i>p</i> <0.001.</p

Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.

<p>Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.</p

Identification of G-quadruplex forming sequences in three manatee papillomaviruses

<div><p>The Florida manatee (<i>Trichechus manatus latirotris</i>) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.</p></div

Hydrodynamic properties of the major TmPV sequences^*.

<p>Hydrodynamic properties of the major TmPV sequences^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195625#t002fn001" target="_blank">*</a>}.</p

Oligodeoxynucleotides used in this study.

<p>Oligodeoxynucleotides used in this study.</p

Molecular dynamics-derived model of the four stacked contiguous G-quadruplexes for sequence TmPV4-2.

<p>Individual G-quadruplex structures appear from top to bottom in orange, magenta, yellow, and red. G-tetrads are identified in elemental colors in Chimera <b>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195625#pone.0195625.ref088" target="_blank">88</a>]</b>.</p

Identification of G-quadruplex forming sequences in three manatee papillomaviruses - Fig 2

<p><b>Coding and non-coding regions aligned across TmPV1 (outer), TmPV3 (middle), and TmPV4 (inner) to illustrate G4 sequences occurring at the same location within regions on the forward (left) and reverse (right) DNA strands.</b> Blue arrows indicate G4 sequences at the same location on TmPV3 and TmPV4. Red arrows indicate G4 sequences at the same location on all three genomes.</p