Aggregation Propensities of Herpes Simplex Virus-1 Proteins and Derived Peptides: An In Silico and In Vitro Analysis.

Recurrent infections of neurotropic herpes simplex virus-1 (HSV-1) have been implicated in etiology and pathology of Alzheimer's disease (AD). Although protein and peptide aggregation events are at the center of the AD pathophysiology, except a single study where a peptide derived from glycoprotein B of HSV-1 was reported to form β-amyloid-like aggregates, similar investigations with the entire proteome of HSV-1 have not been attempted. In the current study, 70 HSV-1 proteins were screened using bioinformatics tools to identify aggregation-prone candidates. Thereafter, the 20S proteasome cleavage sites within the sequence of the selected proteins were determined using Pcleavage and NetChop algorithms, thereby mimicking a cellular proteasomal activity providing short peptides. Here, we report the biochemical characterization of a 28-residue-long peptide (HSV-1 gK208-235) derived from glycoprotein K of HSV-1. The peptide showed high aggregation propensity and homology to the C-terminus of Aβ1-42 peptide. The aggregates of gK208-235 peptide were characterized by the Congo red and Thioflavin T assays and Fourier transform infrared (FTIR) spectroscopy, and their spheroid oligomeric structure was established by atomic force microscopy (AFM). Furthermore, the aggregates demonstrated dose-dependent cytotoxicity to primary mouse splenocytes. The current findings hypothesize a mechanism by which HSV-1 may contribute to AD, which may be pursued further in the future.

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the presence of Aβ1–42 amyloid plaques in the brain and tangles of tau protein inside the neuron cells, which lead to progressive dementia and finally death. Among various environmental risk factors, persistent infection of brain cells by bacteria and virus, especially herpes simplex virus type-1 (HSV-1), has been observed to play a role in AD.[1−5] The presence of HSV-1 in association with amyloid deposition in the cerebral cortex region of the brain was established more than a decade ago in patients with familial AD.[6] HSV-1 was also shown to upregulate Aβ generation in cultured neuronal and glial cells,[7] and later its genomic DNA was shown to colocalize with amyloid plaques found in the brain of patients with AD.[8] A few other studies have implicated this virus for increased risk of dementia, disruption of genetic and molecular networks,[9] seeding β-amyloidosis in brain cells,[10] and AD-specific phosphorylation of tau protein.[11] The data discussed so far have correlated HSV-1 infection and AD comprehensively; however, mechanistic events still remain poorly understood. Even though a peptide derived from glycoprotein B of HSV-1 has been reported earlier to form β amyloid-like aggregates,[12] the aggregation potential of HSV-1 proteome has not been analyzed to date. The current study adopts a hypothetical cellular proteasomal activity as the basis for the generation of HSV-1 peptides, as the proteasomes, in addition to maintaining cellular protein turnover, are also involved in generating peptides from viral proteins, which are then presented on the cell surface of T-cells[13] and demonstrate the aggregation properties of one such peptide in silico and in vitro. The sequences of HSV-1 proteins were retrieved from online databanks and screened for identifying aggregation-prone proteins using software TANGO and AGGRESCAN. Further, the 20S proteasome cleavage sites within the selected proteins were predicted using an in silico support vector machine (SVM) method using an online server. The aggregation-prone peptides were identified using the data of 20S proteasome cleavage sites and analysis of the hydrophobic regions present along the entire length of the selected proteins (gM and gK). These studies led to the identification of a peptide (208LYHRPAIGVIVGCELMLRFVAVGLIVGT235) derived from HSV-1 glycoprotein K (HSV-1 gK208–235), which showed the aggregation score and hydrophobicity value equivalent to those of the Aβ1–42 peptide. Additionally, the sequence comparison of HSV-1 gK208–235 and Aβ1–42 peptide revealed homology at their C-termini. Thereafter, the synthetic peptide was subjected to in vitro solubilization and the resultant aggregates were characterized using Congo red and Thioflavin T (ThT) fluorescence assays and Fourier transform infrared (FTIR) spectroscopy. All of these studies suggested the formation of amyloid-like aggregates. The overall shape of the peptide aggregates was found to be spheroid instead of classical fibrillar structures, as revealed by atomic force microscopy (AFM). The cytotoxicity of the spheroid aggregates was assessed via cell viability assay performed using primary cells—mouse splenocytes, wherein the dose-dependent toxicity was observed.

Results

In Silico Screening for Selection of Aggregation-Prone HSV Proteins

The average aggregation scores of 70 proteins of HSV-1 [Supporting Information (SI), Table S1], FMRP-1 (Fragile-X-Mental Retardation-1 Protein), low aggregation-prone protein (negative control), and a well-established amyloidogenic peptide, Aβ1–42 (positive control) were calculated using software TANGO and AGGRESCAN. FMRP-1 was chosen as a negative control protein as it is highly abundant in neuron cells and plays a role in synapse, cell-to-cell communication,[14] and is not known to have amyloidogenic properties. On the other hand, the Aβ1–42 peptide was chosen as the positive control peptide as its amyloidogenic aggregation properties are well characterized in the literature and is known to form amyloid fibrils.[15,16] Therefore, the protein/peptide, which showed an aggregation score close to that of the Aβ1–42 peptide, could be considered as aggregation-prone candidates. The average aggregation score of each protein is calculated by summing up the aggregation scores of each residue (total aggregation score) of the protein and dividing it by the total number of residues in the protein or peptide. The TANGO/AGGRESCAN average aggregation scores of all proteins were compared to the score of negative control protein FMRP-1 (score of 2.59/0.6) and the positive control peptide Aβ1–42 (score of 36.48/3.6). Software TANGO and AGGRESCAN both identified glycoprotein M and K as the most aggregation-prone proteins, as suggested by their highest average scores (SI, Table S1). As shown in Figure A, the gM and gK showed TANGO average aggregation scores comparable to that of the peptide Aβ1–42, whereas in the case of AGGRESCAN scores, the values of gM and gK were found to be more than 3 times higher than those of the Aβ1–42 peptide. Hence, these two proteins were chosen for further analysis. It is important to note here that the shorter length of the Aβ1–42 peptide may influence its score; therefore, to neutralize this bias and compare the aggregation properties of control and test subjects more explicitly, the aggregation scores, which are calculated per residue across the entire length of the proteins, were determined using TANGO and AGGRESCAN. As shown in Figure B, both gM and gK demonstrated several intermittent segments displaying aggregation scores equivalent to (TANGO) or more (AGGRESCAN) than Aβ1–42 peptide, whereas the negative control, FMRP-1, exhibited a minimum number of such regions, which were of smaller lengths and aggregation scores. This analysis further reaffirms the high inherent aggregation propensity of the selected proteins.

Figure 1

Aggregation and average aggregation scores of HSV-1 proteins gK and gM, Aβ1–42 peptide, and FMRP-1. (A) Average aggregation score of HSV-1 proteins gK and gM, positive control peptide Aβ1–42, and negative control FMRP-1 as determined using software TANGO and AGGRESCAN. (B) Graphical representation of aggregation scores per residue across the entire length of the test subjects. In the description of proteins, the last digits indicated after the names of the proteins/peptide depict the length of the respective proteins.

Prediction of 20S Proteasome Cleavage Sites and Identification of Aggregation-Prone Peptide(s)

The 20S proteasome cleavage sites present in the glycoprotein M and K were predicted in silico by the Pcleavage, an SVM-based method, and NetChop algorithms. According to the obtained data, the majority of the predicted cleavage sites in the proteins were positioned within the regions identified as aggregation-prone (SI, Table S2), leaving a few such regions intact. The aggregation-prone regions in gM and gK (identified using TANGO) that were located between two consecutive 20S cleavage sites were considered for peptide selection. Across the entire length of gK and gM, seven and eight such regions were observed, respectively, as shown in SI Table S2. Analysis of sequences of all of the 15 aggregation-prone regions of gM and gK combined and the positions of the cleavage sites (predicted by Pcleavage) revealed only one continuous peptide, a 28-residue-long stretch of gK (HSV-1 gK208–235). The 20S cleavage sites, which were predicted using the NetChop algorithm, had placed two more sites in the middle of the HSV-1 gK208–235 peptide region, producing two peptides out of the same regions, but of much shorter length. Between the two prediction tools, Pcleavage and NetChop, the former has been reported to predict cleavage sites with a higher accuracy;[17−19] therefore, peptide HSV-1 gK208–235, generated using the Pcleavage algorithm, was chosen for further analysis. The average aggregation score and hydrophobicity values of the Aβ1–42 and HSV-1 gK208–235 peptides were recalculated using the TANGO/AGGRESCAN algorithms and peptide analyzing tool (Thermo Fisher), respectively, and compared. The hydrophobicity value of the HSV-1 gK208–235 peptide (58.68) was found to be slightly higher than that of Aβ1–42 peptide (54.77), as shown in Figure A. The TANGO average aggregation score of HSV-1 gK208–235 peptide (32.58) was found to be equivalent to that of the Aβ1–42 peptide (36.48), with the latter having a slightly higher value (Figure B). In contrast to this, the AGGRESCAN average aggregation score of HSV-1 gK208–235 peptide (12.33) was found to be slightly higher than 3 times the value of the Aβ1–42 peptide (3.61), as shown in Figure B, indicating a high propensity of the test peptide for aggregation. A sequence analysis of these peptides revealed that the C-terminal sequences of both HSV-1 gK208–235 (22VGLIVG27) and Aβ1–42 (32IGLMVG37) peptides were homologous to each other (Figure C). Additionally, the presence of a cysteine residue in the middle of the HSV-1 gK208–235 peptide may form disulfide bonds among aggregated species, further stabilizing the aggregates. The Aβ1–42 and HSV-1 gK208–235 peptides were also analyzed using AMYLPRED2, a web tool that uses a consensus of different methods, which are developed to predict the amyloidogenic features of protein/peptide. The output of the program revealed that, in the case of the HSV-1 gK208–235 peptide, 23 out of 28 residues showed amyloidogenic propensity, a tally higher than the positive control Aβ1–42 peptide (SI, Figure S1), suggesting the potential of the test peptide to form amyloidogenic aggregates. The glycoprotein K was also examined for the presence of glycosylation sites across its primary sequence using UniProt database (P68333). The only two residues at positions 48 (Asparagine) and 58 (Asparagine) were reported to be glycosylated, which did not fall within the sequence of the HSV-1 gK208–235 peptide, further suggesting that the behavior of its aggregation will not be affected by glycosylation. These in silico analyses of the HSV-1 gK208–235 peptide suggested that it has amyloidogenic properties and therefore it was selected for further in vitro studies.

Figure 2

(A) Graphical comparison of hydrophobicity of the Aβ1–42 and HSV-1 gK208–235 peptides. (B) The predicted TANGO and AGGRESCAN average aggregation scores of Aβ1–42 and HSV-1 gK208–235 peptides. The HSV-1 gK208–235 peptide demonstrates comparable aggregation (TANGO and AGGRESCAN) values to that of the Aβ1–42 peptide. (C) Graphical representation of TANGO and AGGRESCAN aggregation scores per residue of Aβ1–42 and HSV-1 gK208–235 peptides. The C-termini of the two peptides showed equivalent aggregation scores, and the homologous sequences toward the C-terminal of Aβ1–42 (32IGLMVG37) and HSV-1 gK208–235 (22VGLIVG27) peptides have been enlarged and underlined.

In Vitro Aggregation Analysis

The peptide HSV-1 gK208–235 was solubilized in the buffer and allowed to aggregate during in vitro experiments. Samples of the peptide were prepared at 55, 100, 200, 300, and 600 μM concentrations in phosphate-buffered saline (PBS) for the aggregation analysis, as described in the Materials and Methods section. The lowest concentration of 55 μM was used in the assay because lower than 50 μM peptide concentrations did not produce enough detectable signals.[20] Second, since the aggregation samples were required to be diluted to a final concentration of 50 μM for fluorescence emission recording, a slightly higher concentration of 55 μM was used as the minimum peptide concentration to maintain the uniformity among the tested samples. The solutions of the peptide were observed to turn turbid instantaneously upon dilution with PBS at all of the five tested concentrations, suggesting thereby a strong propensity of the HSV-1 gK208–235 peptide to aggregate. The sample prepared at a 100 μM peptide concentration was analyzed for the presence of amyloid aggregates by Congo red assay. As shown in Figure , the sample produced a characteristic red bathochromic shift in the absorption maxima of Congo red dye, from 483 to 507 nm. This 24 nm shift in Congo red absorption maxima indicated the possibility of the presence of amyloid-like aggregates. To confirm this nature of the aggregates, the samples were further subjected to amyloid-specific ThT fluorescence assay.

Figure 3

Congo red absorption spectrum of the peptide HSV-1 gK208–235 aggregate showing the characteristic red bathochromic shift in the absorption spectra from 483 to 507 nm in 1 × PBS, pH 7.4. This shift in absorption spectra is indicative of the presence of amyloid-like aggregates.

The peptide samples prepared at 55, 100, 200, 300, and 600 μM concentrations were mixed with the ThT dye, as explained in the Materials and Methods section. The resultant samples were excited at 450 nm, and the emission scan was recorded at 470–700 nm with the peak intensity at 485 nm. As shown in Figure A, a concentration-dependent increase in the fluorescence signal was recorded with peptide samples prepared at 55, 100, and 200 μM; however, the increment in the fluorescence signal, obtained with the sample prepared at 200 μM concentration, was not similar to what was observed with the sample prepared at the 100 μM peptide concentration. This suggested the occurrence of a phenomenon of self-association among the initially formed aggregates, wherein self-association reduces the number of sites available for ThT dye binding and hence results in decreased fluorescence emission. As shown in Figure A, the fluorescence signals remained almost similar for samples prepared at 100, 200, and 300 μM, suggesting the self-association events to be the reason behind the observation. Likewise, the fluorescence emission of the sample prepared at 600 μM concentration reduced further, demonstrating the increased association of aggregates at such a high concentration (Figure A). Such behavior of self-association has been reported earlier with the amyloid fibrils at high peptide concentrations.[21] These observations indicate the possible presence of amyloid-like aggregates in the samples. Further, the kinetics of the peptide aggregation was studied over a period of 48 h using samples prepared at 100 μM peptide concentration. After setting up the aggregation reaction, the samples were withdrawn at 0 h and thereafter after every 4 h, up to 16 h, and the latter samples were withdrawn at 24 and 48 h. The diluted samples were mixed with the ThT dye, and an emission scan was recorded as described earlier. Since the peptide showed instant aggregation, the maximum fluorescence intensity was recorded at 0 h. Thereafter a decrease in fluorescence intensity was recorded up to 24 h. The fluorescence intensity was observed to have stabilized thereafter as the emission graph of the 48 h sample almost overlapped with the 24 h sample (Figure B). A similar behavior of instant aggregation and a subsequent decrease in fluorescence intensities have been reported earlier. Cloe et al. (2011) showed that a shorter mutant peptide ΔE22-Aβ1–39, generated from a mutant ΔE693 (Japanese) β-amyloid precursor protein, aggregates instantly and also forms amyloid fibrils.[21] In another recently reported study, Adler et al. (2017) enhanced the hydrophobicity of the Aβ1-40 peptides by chemically modifying their N-terminus with saturated octanoyl or palmitoyl lipid chains. The lipid modification increased the local hydrophobicity of the peptide, which led to the acceleration in fibrillation kinetics.[22]

Figure 4

ThT fluorescence assay with the peptide HSV-1 gK208–235 in 1 × PBS, pH 7.4. ThT dye binds to amyloid aggregates and fluoresces at 485 nm. The results are expressed as mean ±SE, and the asterisk (*) denotes the statistical significance with P-value < 0.05. (A) Fluorescence emission analysis of HSV-1 gK208–235 peptide aggregates prepared at different peptide concentrations (55–600 μM). A significant increase in ThT fluorescence was observed after binding to HSV-1 gK208–235 peptide aggregates, suggesting their possible amyloidogenic nature. The peptide samples were analyzed after 24 h of incubation. (B) Kinetics of HSV-1 gK208–235 aggregation, set at a concentration of 100 μM. The ThT fluorescence was recorded to be highest at 0 h, which thereafter declined up to a time-lapse of 24 h. The ThT fluorescence stabilized thereafter and did not show further decline even at 48 h.

The aggregation sample prepared at a 100 μM peptide concentration was further analyzed to determine the structural features of the peptide aggregates. The sample was prepared as described in the Materials and Methods section and subjected to AFM. The peptide aggregates were observed to form spheroid oligomeric species of diameter ∼15 nm (Figure A). Further, the z (axis) profile analysis revealed the height of the oligomer as ∼12 nm, as depicted in Figure B. No classical amyloid fibrils were observed in the AFM analysis.

Figure 5

Atomic force microscopy (AFM) of aggregates of the HSV-1 gK208–235 peptide. (A) AFM image of aggregates showing the presence of spheroid oligomers with a 200 nm scale bar. The inset picture is the magnified image of spheroid oligomers. (B) Graph is depicting the z profile of the spheroid oligomers.

Secondary Structure of Peptide Aggregate

To further affirm if the spheroid oligomeric aggregates contain amyloid-like structures, the aggregation sample was subjected to attenuated total reflection (ATR)-FTIR spectroscopy. The analysis of FTIR spectra allows the prediction of the protein secondary structure content. The infrared spectrum of a protein often contains peaks of multiple amide bonds due to the vibrational contributions from the amino acid side chains and protein backbone. The absorption band generated by C=O stretching in the peptide is designated as amide I, and the N–H banding pattern is denoted as amide II. The amide I band in the spectra is useful in predicting the secondary structure of protein/peptide.[23] The amide bands at 1634/1635 cm–1, 1645 cm–1, and 1651/1653/1655 cm–1 correspond to the β-sheet, random coil, and α-helix secondary structures, respectively.[24,25] The FTIR spectra of spheroid aggregate showed a sharp band at 1634.84 cm–1 (Figure ), indicating the presence of a high β-sheet content. The wide band between 3100 and 3500 cm–1 shows the presence of residual moisture (H2O) in the peptide aggregate. A highly symmetrical amide I band made it difficult to process spectra for deconvolution to estimate the random coil/α-helix in the spheroid aggregate. This symmetrical and prominent amide I band at ∼1634 cm–1 suggests the presence of a high β-sheet content in the spheroid oligomer, further indicating the formation of amyloid-like aggregates.

Figure 6

Attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR) transmittance spectra of the spheroid HSV-1 gK208–235 peptide aggregates. The sharp and prominent amide I band at 1634 cm–1 indicated the presence of β-sheet rich structures in the spheroid peptide aggregates.

Cytotoxic Properties of HSV-1 gK208–235 Peptide

Accumulation of the neurodegenerative plaques, composed of Aβ1–42 peptide, is the most prominent of all pathological hallmarks observed in the brain of patients with AD. The toxic effects of Aβ1–42 amyloid aggregates have been established in neurons and many other cells when applied extracellularly.[26,27] To determine if HSV-1 gK208–235 amyloid-like spheroid aggregates were cytotoxic, a preliminary cell viability assay was performed using mouse primary cells. The splenocytes were isolated from mouse spleen and were grown in culture in the presence of viral peptide aggregates at concentrations 2.5, 5, 10, and 20 μM for 48 h. The estimation of viable cells was performed at 24 and 48 h after culture, as described in the Materials and Methods section. Although untreated cells appeared healthy up to 48 h with a negligible reduction in viable cell percentage, dose-dependent cell death was observed in treated cells, wherein extensive cell death was observed at 10 μM and higher concentrations of aggregates after 48 h of culturing (Figure A,B). The median toxic dose (TD50) of the HSV-1 gK208–235 peptide aggregates against the mouse primary splenocyte cells was observed at 7.11 and 4.35 μM for 24 and 48 h, respectively. Collectively, the in silico and in vitro results suggest that the HSV-1 gK208–235 peptide is capable of self-assembly into spheroid oligomeric amyloid-like aggregates that are toxic to primary cells. Hence, we hypothesize that primary or recurrent infections of HSV-1 may lead to the release of HSV-1 gK208–235 and/or other such peptides inside host cells, leading to the formation of amyloid-like aggregates, and prove toxic to neurons or other host cells.

Figure 7

(A) Pictures in the grids captured by inverted phase-contrast microscopy of the primary splenocyte cells. Control (column 1) and treated cells (column 2–4) with different concentrations (2.5, 5, 10, and 20 μM, respectively) of HSV-1 gK208–235 peptide aggregates showing distinct cytotoxic morphological changes in a dose-dependent manner. The peptide aggregates in the pictures are indicated by the black arrows. (B) Representative data in the bar graph showing observed cell death percentage in primary splenocyte cells estimated by trypan blue exclusion assay. The results are expressed as mean ± SE, and the asterisks (*) denote the statistical significance with P-value < 0.05.

Discussion

Herpes simplex virus-1, a neurotropic virus, following its primary infection establishes a latent infection in sensory ganglia, especially the trigeminal, through retrograde axonal transport.[28] The recurrent activation of HSV-1 may allow it to enter the central nervous system (CNS) in either manifesting a disease condition, for example, herpetic encephalitis, or entering latency.[29,30] By employing CD8+ T cells, which recognize viral antigen peptides presented on the infected cell surface in complex with major histocompatibility complex-1 (MHC-1) molecules, the immune system effectively eliminates virus-infected cells and keeps viral spread under check. In relation to antigen presentation in the CNS, the expression of MHC-I molecules in the hippocampus area of the human brain, endothelium and microglia, was reported long before it was observed in patients with AD.[31] According to the earlier belief, neurons were devoid of major histocompatibility complex-1 (MHC-1) expression and were considered immune-privileged.[32] However, many studies have shown the expression of MHC-I molecules in neurons of the human brain.[33−35] Additionally, the potential of the neuroinflammatory machinery is well studied in a neurodegenerative disease like Parkinson’s disease (PD).[36] According to a recent study, the recurrent infection of HSV-1 in mice was shown to induce hallmarks of neurodegeneration as observed in AD, e.g., accumulation of Aβ protein, hyperphosphorylation of tau protein, and induction of neuroinflammation.[37] The periodic reactivation of HSV-1 suggests its effective evasion of the immune system at least in the initial stages of the infection. Since the presentation of peptides with MHC-I is the key step of antigen presentation and is linked to cytotoxic cell death,[38] blocking antigen presentation to cytotoxic T lymphocytes (CTL) helps the virus to remain hidden. In cells including neurons, peptides used for antigen presentation are primarily released by the 20S proteasome activity. The proteases and peptidases located in the cytoplasmic and endoplasmic reticulum (ER) then trim these larger peptides at the amino terminus for MHC-1 presentation.[39,40] These antigenic peptides are transported across the ER through a transporter associated with antigen processing (TAP). Inside the ER, these peptides are further trimmed into shorter peptides by peptidases and loaded on to MHC-1 molecules for surface display.[41−45] Under normal physiological conditions, this mechanism leads to the recognition of virus-infected cells by the host immune system, especially for CTL responses. In 1995, two research groups demonstrated separately that HSV-1 evades host immune response by blocking the MHC-1 presentation of its antigenic peptides.[46,47] Both the groups demonstrated that infected cell protein-47 (ICP-47), encoded by HSV-1, inhibits the transport of peptides across the ER, leading to the inhibition of MHC-1-mediated antigen presentation. HSV-2 has also been reported to encode ICP-47 variant protein with the same function.[48] Recently, a report demonstrated that a conserved sequence, “50PLL52”, present within the central region of ICP-47 is essential for its inhibitory activity.[48] These earlier findings and the observations made with the HSV-1 gK208–235 peptide in the current study led us to hypothesize that blocking of TAP by HSV-1-encoded ICP-47 may create a transient accumulation of HSV-1-derived peptides in addition to cellular peptides. This milieu of peptides of HSV-1 origin generated by 20S proteasome activity, which may include HSV-1 gK208–235 as well as other peptides of HSV-1, if not during every occasion of reactivation, at some point of time may contribute to intracellular aggregation events. The hypothesis is schematically depicted in Figure . The HSV-1 gK208–235 peptide, which was identified in the current study, showed self-assembly into spheroid and cytotoxic aggregates.

Figure 8

MHC class I antigen presentation pathway and mechanistic hypothesis of the generation of amyloidogenic like cytotoxic aggregates of HSV derived peptides. Viral proteins are proteolytically processed in the cytosol by 20S proteasome. The peptide fragments generated by the proteasome are translocated into the ER lumen by transport-associated protein (TAP) for further trimming and binding MHC-1 molecules. The peptide fragment-loaded MHC class I molecules are transported through the secretory pathway to the plasma membrane for recognition by cytotoxic CD8+ cells. The herpesvirus encodes for an infected cell protein-47 (ICP-47). The ICP-47 has been shown to interfere in peptide transport from the cytosol to ER. This blockage of peptide transport across the ER may trigger the aggregation of peptides of varying hydrophobicity to form intracellular amyloidogenic like cytotoxic aggregates.

Peptide HSV-1 gK208–235 showed aggregation propensities equivalent to control Aβ1–42 peptide even though it is half the size of the latter. In addition to showing equivalent scores of aggregation and hydrophobicity, the two peptides also share a short homologous sequence of six amino acid residues at their respective carboxyl termini. The abundance of hydrophobic amino acid residues across the entire length of the HSV-1 gK208–235 peptide, as reflected in its aggregation parameters, makes it prone to instant aggregation. This behavior of the peptide was clearly reflected during in vitro solubilization studies aimed at analyzing the kinetics of its aggregation. The maximum aggregation, measured in terms of ThT fluorescence intensity, was obtained at 0 h, which suggests that the monomeric peptide, dissolved in dimethyl sulfoxide (DMSO), upon diluting with aqueous buffer aggregated instantly. The samples, collected up to 48 h, showed a decline in fluorescence up to 24 h and got stabilized thereafter. This observation is indicative of a dynamic aggregation process undergoing continuous peptide association and dissociation and stabilizes upon reaching an equilibrium-like state, as was observed with the stabilization of the ThT fluorescence. The C-terminus of the HSV-1 gK208–235 peptide is homologous to the C-terminus of the Aβ1–42 peptide (Figure B). An N-terminal truncated Aβ1–42 peptide has been shown to form spherical oligomeric channels instead of amyloid fibrils, and this form of aggregates also has been found to be neurotoxic.[49] The truncated Aβ1–42 peptide is of the length equivalent to that of the HSV-1 gK208–235 peptide. The similarity between these two peptides at the level of length, sequence, and shape of their amyloid-like spheroid aggregates suggests a similar function for the HSV-1 peptide. For performing cytotoxicity experiments, aggregates of the HSV-1 gK208–235 peptide were generated at a 100 μM concentration, and thereafter, diluted samples were added to the splenocyte cultures. The fact that the peptide is highly prone to aggregation is shown by the kinetics of its aggregation, and its presence in free form is highly unlikely and it was assumed that the aggregation samples contained a negligible amount of free peptide. The AFM data had shown the structure of the aggregates to be nonfibrillar and of spheroid shape. A recent study, where the oligomeric species were found to be more toxic compared to the fibrils,[50] suggests that nonfibril structures are potent toxins. Additionally, the aggregates showed concentration-dependent cytotoxicity, even at aggregate concentrations as low as 2.5 μM, indicating the role of aggregates in the observed cytotoxicity. Although the actual mechanism of action of these aggregates remains to be elucidated and may be pursued in future studies, it may be noted that spherical aggregates were prepared at 100 μM (∼0.33 mg/mL); however, their cytotoxicity was observed at a much lower concentration (<10 μM). The 10 μM peptide concentration is equivalent to 0.03 mg/mL, which is much lower than that of the cytoplasmic protein concentration, estimated to be ∼100 mg/mL,[51] and the total macromolecular concentration, including proteins, lipids, and sugar, of ∼400 mg/mL.[52] However, the crowded environment of the cell cytoplasm may provide a condition where the effective concentration of the freshly released peptides, after 20S proteasome cleavage, may remain high due to low diffusion amidst crowding and aided by their blocked transport into the ER lumen. These events may not be common under normal conditions, but may arise during HSV-1 infection and may lead to aggregation events at low peptide concentrations. Once the aggregates are formed, they were found toxic at very low concentrations, as shown in our study.

Additionally, according to one report, proteins that are involved in neurodegenerative disease were found more likely to form amyloid fibrils under crowded conditions than in dilute solutions.[53] However, the effect of such molecular crowding on the HSV-1 gK208–235 peptide aggregation needs to be pursued further.

We would like to link hypothetically another hallmark event that is observed in AD pathology, which is hyperphosphorylation of tau protein with HSV-1 recurrent infection.[54] The HSV-1 infection has been shown to contribute directly to the hyperphosphorylated forms of tau, which are extremely prone to aggregation.[11] Hence, HSV-1 during active infection is likely to express the protein ICP-47, which may block the TAP-facilitated transport of peptides across ER, and simultaneously HSV-1 kinase may phosphorylate tau protein making it prone to aggregation. The intraneuronal buildup of aggregation-prone viral peptides and hyperphosphorylated tau protein may trigger the aggregation process.

With the background knowledge about established facts of HSV-1 infection and AD pathology and the observations made during the current study, we would like to propose a theory that HSV-1 peptides, which may be generated during HSV-1 infection due to proteasomal activity, may contribute to AD etiology and pathology in combination with other events triggered by the viral infection. This mechanistic hypothesis offers a fresh look at the events that follow an HSV-1 infection and may play a combined role in AD; although, the hypothesis still remains to be validated by more substantial in vitro and in vivo support.

Materials and Methods

Protein Sequences

Protein sequences of HSV-1 were retrieved from the NCBI protein database (http://ncbi.nlm.nih.gov/protein/) and UniProt (http://uniprot.org/) using keywords like human herpesvirus 1, envelope glycoproteins, or by the respective protein name. The protein sequence of Fragile-X-Mental Retardation-1 Protein (FMRP-1), an abundant and constitutively expressed protein in normal healthy brain neurons,[55] (UniProt, Q06787), and a well-established amyloidogenic peptide, Aβ1–42,[15] were used as negative and positive controls, respectively, for in silico aggregation prediction.

Prediction Tools

Online software TANGO (http://tango.crg.es/), AGGRESCAN (http://bioinf.uab.es/aggrescan/), and AMYLPRED2 (http://aias.biol.uoa.gr/AMYLPRED2/), based on computer algorithms, were used for the prediction of aggregation-prone regions in unfolded polypeptide/peptide chains.[56−59] For predicting the proteasome cleavage sites of proteins, Pcleavage, a support vector machine (SVM)-based method (http://www.imtech.res.in/raghava/pcleavage/index.html), and NetChop V3.0, based on artificial neural network (http://www.cbs.dtu.dk/services/NetChop/), were used.[17,18] The hydrophobicity of the peptide fragments was calculated using an online peptide analyzing tool (Thermo Fisher).

Prediction of 20S Proteasome Cleavage Sites

The retrieved protein sequences were scrutinized for aggregation score using software TANGO and AGGRESCAN at default parameter values, viz., pH 7.4 and 310 K temperature. No sliding window was used for the TANGO and AGGRESCAN score calculation. Selected glycoproteins of HSV-1 were fed into the proteasome cleavage prediction tools Pcleavage and NetChop with threshold/cutoff values of 0.6 and 0.9, respectively.

Aggregation Sample Preparation

A 28-residue-long peptide fragment (208LYHRPAIGVIVGCELMLRFVAVGLIVGT235) derived from HSV-1 glycoprotein K was chemically synthesized in the lyophilized form with >95% purity (“S” BioChem, India). The 1 mg peptide was dissolved in a small amount (∼25 μL) of dimethyl sulfoxide (DMSO) and diluted with Milli-Q water to prepare a stock solution of 1 mg/mL such that the concentration of DMSO remains less than 3%. The stock solution was then centrifuged at 15 000g for 2 min. The final concentration of the dissolved peptide in the stock solution was estimated by Bradford’s assay. Aggregation samples were prepared by mixing desired volumes of peptide and phosphate-buffered saline (10 × PBS) stock to obtain desired concentrations, viz., 55, 100, 200, 300, and 600 μM, of the peptide in 1 × PBS. The resultant peptide solutions were incubated on a thermomixer at 37 °C and stirred at 1200 rpm for the desired period. The samples were then analyzed using Congo red absorption and Thioflavin T fluorescence assays.

Congo Red Absorption Assay

The aggregation samples were mixed with Congo red dye (Sigma-Aldrich) for absorption assay. A working solution of 15 μM Congo red was prepared from a stock of (0.1%) 1435 μM stock solution. The aggregation sample (150 μL, 100 μM) was mixed with 3.13 μL of Congo red stock solution in such a way that the final prepared concentration of 50 μM of peptide aggregate and 15 μM Congo red is achieved in a total of 300 μL volume of 1 × PBS. The absorption spectra of the resultant samples were recorded at 400–600 nm wavelength at a resolution of 1 nm using an ELISA plate reader (Thermo Fisher).

Thioflavin T (ThT) Fluorescence Assay

The selective binding of mature amyloid fibrils with Thioflavin T emits fluorescence in the wavelength range of 485–500 nm.[60] Different volumes of samples of aggregation reactions, set at 55, 100, 200, 300, and 600 μM concentrations of peptide, were mixed with the ThT stock solution (10×) in 1 × PBS to obtain a final concentration of 20 μM ThT and a peptide concentration of 50 μM. The resultant samples were excited at 450 nm, and the fluorescence was recorded in the range of 470–700 nm on a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies). The slit width of excitation and emission was 5 nm. The peak intensity of fluorescence at 485 nm was used for statistical analysis (mean values and standard error mean).

Atomic Force Microscopy (AFM)

A custom cut 10 × 10 mm glass slide of thickness 1 mm was treated with a saturated solution of potassium hydroxide in absolute ethanol, as described in ref (61), to minimize the surface roughness. The peptide aggregate was washed three times with Milli-Q water by centrifugation (12 000g for 2 min) and resuspension cycles. The washed peptide aggregate was incubated on a glass slide and dried in a dust-free environment for 10 min, and the images were obtained by AFM scan. The AFM data were analyzed using WSxM software.[62] Atomic force microscopy (AFM) was performed at Material Research Centre, Malaviya National Institute of Technology (MNIT), Jaipur, Rajasthan, India.

ATR-FTIR Spectroscopy

The peptide aggregate was analyzed by attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy. The peptide aggregate sample washed with 10 μL of Milli-Q water was dried on a diamond crystal in an ATR cell. A PerkinElmer Spectrum Two FTIR spectrometer with an MCT detector was used to measure the spectrum in the spectral range of 4000–400 cm–1 at a resolution of 1 cm–1 and an average of 64 scans. FTIR spectroscopy was performed at Material Research Centre, Malaviya National Institute of Technology (MNIT), Jaipur, Rajasthan, India.

Cell Viability Assay

The C57BL/6 mice were kept at the animal house facility of the University of Rajasthan, Jaipur. The splenocytes were gifted by Dr. A. S. Ansari’s lab for the current experiment. The mice were sacrificed, and the spleen was removed aseptically. The isolated spleen was washed in cold PBS. The spleen was teased gently against a sterile frosted glass slide to dislodge the cell mass in RPMI 1640 medium. The resultant splenocyte suspension was washed in PBS and the cells were collected by centrifugation at 200g for 5 min at 4 °C. The pellet obtained was suspended in a red blood cell (RBC) lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM ethylenediaminetetraacetic acid, EDTA) and incubated for 10 min on ice, and the cells were collected by centrifugation at 200g for 5 min. The collected cell pellet was washed again in RPMI 1640 medium and finally resuspended in 2 mL of RPMI 1640 medium with 10% serum. The counting of viable cells was performed by mixing the diluted cell suspension with 0.4% trypan blue in a 1:1 ratio and loading the mixture onto a hemocytometer, wherein the cells were counted using an inverted microscope (Zeiss Primovert). The final splenocyte count was adjusted to 1 × 106 cell/mL. The splenocyte (0.5 mL) in RPMI 1640 medium with 10% serum was added to the wells of 24-well culture plates with different concentrations (2.5, 5, 10, and 20 μM) of peptide aggregate and incubated in a 5% CO2 incubator at 37 °C up to 48 h. The peptide aggregates were prepared at a concentration of 100 μM, as described earlier. The images of the cells were captured by a Zeiss Primovert inverted microscope at a final magnification of 400×, and the counting of viable cells was performed by mixing the cell suspension with 0.4% trypan blue at a 1:1 ratio.

Statistical Analysis

The statistical significance was accepted at P < 0.05. Analysis of variance (ANOVA) test was used for statistical analysis. Statistical analyses were performed using GraphPad Prism 6.0 software.