Introduction
Monkeypox virus is a double-stranded DNA virus and one of the human pathogenic orthopoxviruses that include
Variola (VARV), cowpox (CPX), and
Vaccinia (VACV) viruses. The virus causes a disease that manifests similarly to smallpox, but with milder morbidity and lower mortality rates [
1]. Variation in MPV virulence has been observed and mapped to defined geographic origins, e.g., virus isolates from Central Africa are more virulent than those from Western Africa [
2,
3]
Recent advances in molecular biology and genomics have improved our understanding of viral infection and replication mechanisms. Monkeypox virus has a relatively large genome of about 196,858 base pairs, encoding 190 open reading frames, which constitute the bulk of the material needed for viral replication in cell cytoplasm [
4]. Viral entry into cells is dependent on cell type and viral strain, and occur after an initial attachment to cell surface through interactions between multiple viral ligands and cell surface receptors [
5] such as chondroitin sulfate [
6] or heparan sulfate [
7,
8]. Subsequent crossing of cell membrane is mediated by a viral fusion event with cell membrane under neutral pH conditions [
9], or by endosomal uptake via a macropinocytosis-like mechanism that involves actin [
10,
11] and low pH-dependent steps [
12]. Once in the cell cytoplasm, the virus releases prepackaged viral proteins and enzymatic factors that disable cell defenses and stimulate expression of early genes [
13‐
15]. Synthesis of early proteins promotes further uncoating, DNA replication, and production of intermediate transcription factors. In following stage, intermediate genes are transcribed and translated to induce the expression of late genes that function mainly as structural proteins, enzymes, and early transcription factors. Eventually, membrane structures will appear and unit virion genomes processed from DNA concatemers are assembled into nascent virions that contain all enzymes, factors, and genetic information needed for a new infectious cycle.
The detailed available information about viral gene functions and its programmed expression during infection exceeds current knowledge of corresponding events in the host. Furthermore, although poxviruses are considered one of the most self-sufficient viral families, they remain unable to reproduce in extracellular environment and known to have limited host range, which suggest dependence on host elements [
16,
17]. Therefore, identification of these specific host elements and pathways that are essential for viral replication will enrich our knowledge of host response to viral infection, and may prove valuable in identifying potential targets for antiviral therapies.
Microarrays have been used in genome exploration and profiling with special focus on understanding dynamics of viral gene expression and pathogenesis [
18,
19]. However, a paucity of work employed this tool in examining host response to infections with poxviruses generally [
20‐
22], and more specifically in the case of MPV. Because combining microarray technology with modern data mining tools allows further information extraction at genome-wide levels, we used whole genome rhesus macaque microarrays in combination with Ingenuity Pathways Analysis (Ingenuity
® Systems,
http://www.ingenuity.com) to investigate the effect of MPV infection on host
Maccaca mulata kidney epithelial cells transcriptome, and address gaps in host response during MPV infection. Functional and canonical pathway analysis of differentially expressed genes at 3 and 7 hours post-infection (hpi) time points validated many of the known host gene responses to poxvirus infection and introduced new sets of interesting functions and pathways in areas of cell death and apoptosis, actin dynamics, ion channels and transport, and cell cycle regulation. Our data points to a vital role for these cell functions in MPV infection, and hence, signify their value in poxviruses' infection diagnosis and treatment studies.
Materials and methods
Cell culture and viral infection
Monkeypox virus-Katako Kombe strain (MPV-KK) was propagated in Vero E6 cells maintained in Eagle's Minimum Essential Medium with non-essential amino acids (EMEM/NEAA) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 10 mg/L Gentamycin, 250 μg/L Fungizone, and buffered at pH 7.4 with 10 mM HEPES [
23]. Viral titers were determined by the plaque assay [
24]. As described previously [
25], monolayer of Vero E6 were inoculated with serial dilutions of viral suspension and allowed to adsorb for 30 min at room temperature. The viral inoculate was removed and cells were washed twice with PBS then incubated with culture medium for 5 days. To count plaques, culture medium was removed and cells were fixed and stained simultaneously using 30% formalin, 5% ethanol (vol/vol) solution containing 1.3 g/L crystal violet.
For time points and control samples, MK2 cells were grown in the same culture medium described above but in absence of antibiotics and Fungizon for at least 2 days before infection with MPV. Culture medium was removed and cells were inoculated with crude monkeypox virus-Katako Kombe strain (MPV-KK) at MOI of 3. Virus was adsorbed for 30 min at 37°C with gentle rocking for 15 sec each 10 min, then cells were washed twice with room temperature equilibrated PBS, fresh culture medium was added, and cells were incubated for 3 or 7 hours. Control cells were handled identically except for exposure to virus.
RNA and cDNA preparation, labeling, hybridization, and scanning
RNA was extracted using TRIzol LS (Invitrogen) according to the manufacturer's recommended protocol. Briefly, 200 μl of chloroform (Sigma Aldrich, St. Louis, MI) was added to 5×106 infected and non-infected control cells harvested in 1 ml of TRIzol LS reagent (Invitrogen). Solutions were mixed thoroughly and incubated for 10-15 min at 4°C, then centrifuged for 20 min at 14,000 rpm in 4°C. After centrifugation, the aqueous phase was removed, added to an equal volume of 2-propanol (Sigma-Aldrich). After overnight incubation at -20°C, the mixture was centrifuged for 20 min at 14,000 rpm in 4°C. Supernatant was removed and the RNA pellet was washed with 80% ethanol (Sigma Aldrich), dried, and dissolved in 100 μl of RNase-free water (Ambion, Austin, TX). Possibly present contaminating DNA was eliminated using Turbo DNA-free kit (Ambion), and RNA clean up was performed using RNeasy kit (Qiagen, Valencia, CA). Quality and quantity of RNA was evaluated using Experion automated electrophoresis station and Experion RNA StdSens analysis kit (Bio-Rad, Hercules, CA). Clean sharp peaks representing intact rRNA were confirmed for each preparation by two independent workers.
cDNA was synthesized using One-cycle cDNA Synthesis kit (Affymetrix, Santa Clara, CA) in presence of poly-A RNA controls. Double-stranded cDNA samples were cleaned up and biotin labeling of antisense cRNA was carried out with the IVT Labeling Kit (Affymetrix, P/N 901229). Material was cleaned up then fragmented before hybridization overnight. Microarrays (Affymetrix, P/N 900656) were scanned using the Affymetrix GeneChip scanner following standard Affymetrix protocols [
26] after carrying out all washing and staining steps as recommended by the manufacturer.
Microarray validation by RT PCR
RT PCR was performed utilizing the Superarray human common cytokines panel (PAHS-021E-4, Superarray, Frederick MD, 21703). Reactions were performed according to manufacturer's guidelines for both time points in 10 μl volumes using the 384 plate format that allow gene expression analysis in quadruplicate. Results showed a strong correlation of gene expression levels with that obtained using microarray.
Data analysis
The Affymetrix CEL files were imported into GeneSpring GX software v 7.3.1 (Agilent), which allows multi-filter comparisons using data from different experiments, to perform the normalization, generation of restriction lists and functional classifications of the differentially expressed genes.
Normalization was applied in two steps: i) "per chip normalization" by which each measurement was divided by the 50th percentile of all measurements in its array; and ii) "per gene normalization" by which each treated sample was normalized against its respective control (mock-treated) sample.
The expression of each gene was reported as the ratio of the value obtained relative to the control condition after normalization of the data. Transcripts whose levels reproducibly changed were identified using one-way parametric analysis of variance with a P-value cutoff of 0.05 (false discovery rate of 5%). The changes in transcript levels are expressed as the fold change in signal between control and treated samples.
Two-dimensional clustering was carried out based on samples and genes for visualization and assessment of reproducibility in the profile of the significant genes across biological replicates.
Discussion
Apoptosis is a natural controlled cell death mechanism triggered by diverse stimuli to maintain tissue homeostasis and eliminate abnormal or infected cells [
68]. Because apoptosis represents an important part of antiviral host response, poxviruses developed numerous ways to target it and disrupt its function [
69]. Diverse anti-apoptotic viral strategies are identified in different poxviruses, e.g.,
Molluscum contagiosum virus (MCV) inhibits caspase-8 by expressing MC159 gene, which encodes a protein that binds procaspase-8 and Fas-associated death domain, thereby inhibiting death receptor-induced apoptosis mediated by Fas, TNF, or TRAIL receptors [
70]. MC066 gene encodes a protein with glutathione peroxidase-like function to convert oxygen-reactive species to neutral molecules, hence preventing apoptosis triggered by increased oxidative stress associated with infection [
71]. Little is known about apoptosis in cells infected with MPV, but related orthopoxviruses exhibit clear anti-apoptotic functions. Cowpox virus for instance expresses a protein that can block apoptosis in multiple ways. One of the most potent anti-apoptotic proteins is the cytokine response modifier A (CrmA) which inhibits caspase-8, caspase-10, and blocks garnzyme B-mediated apoptosis [
72,
73]. Similarly,
Vaccinia viruses use SPI-2 family protein member of serine protease inhibitors (serpins) encoded by B13R gene to block apoptosis induced by death receptors [
74]. The same virus inhibits apoptosis induced by RNA-dependent protein kinase (PKR) using specific PKR inhibitors encoded by E3L and K3L genes [
75], and apoptosis induced by loss of mitochondria outer membrane potential by expression of F1L gene [
76].
The presence of many viral proteins that block apoptosis at multiple points suggests that apoptosis is detrimental to viral survival. However, the observed downregulation of Bcl-2, PUMA, and PAK2 and upregulation of NOXA and caspase-3 with the negligible change in other apoptotic genes in our data is more consistent with apoptosis induction. Although many pathogens and viruses other than poxviruses are reported to promote apoptosis [
77], it is unlikely that MPV will differ from other poxviruses in their common anti-apoptotic trend, and the observed divergence between the regulation of apoptosis-specific genes in MPV-infected cells and overall anti-apoptotic outcome seen in other poxvirus-infected cells suggests an anti-apoptotic viral mechanism that functions downstream of apoptosis induction in the host. MPV genes involved in blocking apoptosis remain unknown, but our data suggests the presence of an ortholog(s) of
Vaccinia virus (F1L) gene in MPV, which acts directly on the mitochondria in Bcl-2 like manner.
Double-stranded DNA mammalian viruses have large genomes reaching 289 Kbp or few hundreds of micrometers in length as in the case of fowlpox virus [
78]. Compacting the genome is an indispensible biological task due to the rich negative charge and large size of DNA molecules. In eukaryotes, this was solved by wrapping DNA around a heterooctameric molecule composed of four dimerized, positively charged core histones to form nucleosomes. Further stability is brought about by linker histones that bind DNA laterally. Nucleosomes, in a nucleofilaments form or in a form of higher-order-structure assemblies, chromatin, play an essential role in the regulation of gene expression and chromatin remodeling via acetylation and deacetylation of the N-terminal histone tails protruding from nucleosomes. Viruses exhibit similar architecture in their DNA compaction. Staining vaccinia-infected cells with osmium ammine-SO
2 revealed areas with concentrated viral DNA in two predominant DNA configurations varying depending on the level of viral synthetic activities. Encapsidated genomes exhibited a nucleosome structure like those observed in resting eukaryotes, which was in agreement with biochemical data showing the supercoiled organization of nucleoproteins extracted from Vaccinia viruses [
79]. On the other hand, like active cellular chromatin, non-encapsidated viral genomes exhibited extended DNA features [
80]. Similar variation in DNA density was observed in Herpes simplex viruses (HSV), another double stranded DNA virus, with two well described configurations of euchromatin or heterochromatin that correlated strongly with lytic or latent HSV infection stages, respectively. Generally, little is known about chromatin potential in poxviruses as an antiviral mechanism. Our results showed an interesting downregulation of five essential histone expression regulation factors and five enzymes regulating chromatin dynamics in MPV-infected cells. It is unclear if these results are part of the host response, which include chromatin-mediated silencing of the viral genome and activation of DNA damage [
81], or part of the viral strategies to take over its host. However, our observation predicts an important role for histone expression, histone posttranslational modification, and dynamic exchanges of chromatin in host-poxvirus interactions. Recent work suggested a role for the viral A32L gene of
Vaccinia virus in DNA packaging based on sequence similarities with the product of gene I of filamentous single-stranded DNA bacteriophages and the Iva2 gene of adenoviruses. Both of these genes are ATPases involved in DNA packaging. Additional
Vaccinia genes that map to I6 or I1 telomere-binding proteins are believed to play roles in DNA packaging, because mutants of VACV in either of these two genes fail to exhibit normal DNA packaging at different morphogenesis stages. The sharp upregulation we observed in three out the four core histones, and the striking similarities in DNA compaction architecture observed in eukaryotes and some viruses, with absence of known poxvirus proteins that exhibit histone-like properties makes it tempting to hypothesize a role of host cell histones in viral DNA compaction and nucleosome formation, especially that similar involvement was described recently in simian virus 40 (SV40) DNA compaction [
82]
Our analysis identified ephrin receptor pathway (ERP) as a major influenced pathway in infected cells. This might be due to either increased cell to cell communication by signaling through this receptor tyrosine kinases (RTK) family in response to infection, or to the presence of many pleiotropic genes that are found in ERP, and simultaneously have essential roles in cytoskeleton reorganization or actin polymerization. Intracellular viral motility and morphogenesis of Vaccinia virus into a cell-associated enveloped virion (CEV) and extracellular enveloped virions (EEV) forms are shown to be driven by interactions of host microtubules and
Vaccinia A27L, A17L and A14L genes. Furthermore, egress of
Vaccinia particles in EEV form and direct cell-to-cell virus dissemination is propelled by actin tail formation, which involves the interactions of
Vaccinia transport genes, including A36R, F12L, and host proteins such as Src family kinases (SFK), Nck, WIP, N-WASP, Arp 2/3 to promote actin filaments nucleation. Actin polymerization produces microvilli at cell surface that lift CEV and project it on adjacent cells to finally deliver the virus with minimal exposure to host immune system. Our results confirm regulation of many principal signaling components involved in actin cytoskeletal dynamics, and introduce additional infection regulated genes with functions related to microtubules signaling. This includes intersectin 1 (SH3 domain protein) gene, which encodes a cytoplasmic membrane-associated protein that indirectly coordinates endocytic membrane traffic with the actin assembly machinery, Rho-effector ROCK1 serve a number of key cellular functions, such as morphological differentiation and cell motility which are closely associated with changes in cytoskeletal dynamics [
83]. Additionally, RAS p21 protein activator (GTPase activating protein) [
84], v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog [
85] and SOS2 [
86] are crucial genes in polymerization of actin filaments and cytoskeleton reorganization.
Ion channels represent an intriguing and novel class of genes that were impacted by MPV infection. We identified 10 genes encoding nine ion channels and a transporter that underwent increasing suppression during infection. Most of these channels localize to cell membrane, and collectively contribute to transport of all essential ions involved in maintenance of cell membrane potential and osmolarity homeostasis. While mechanisms of transport modulation have been described previously, as in the indirect consequences of Ras, Rho, and Rab small GTPases regulation [
87], its effect on viral infections and global cell biology remain unclear except for a recent report describing the interaction of myxoma poxvirus protein M11L with mitochondrial permeability transition pore and its role in delaying apoptosis in host cells [
88]. The downregulation trend of channel expression identified here pose many intriguing questions, especially in the light of evolving evidence in support of ion channels role in virus release [
89] and infected cells rupture [
90].
Progression of the cell cycle is tightly regulated process with many redundant checkpoints that ensure proper transition across cell cycle phases. Our results showed significant modulation in the expression of many genes that play essential roles in cell cycle regulation, which led to the identification of ATM signaling, G2/M DNA damage checkpoint, regulation by BTG family protein, and G1/S checkpoint as major influenced pathways during MPV infection. A core cell cycle regulation gene, Cdc25 kinase, has three essential homologs Cdc 25A/B/C that exhibited significant regulation upon MPV infection. While Cdc25B/C showed downregulation favoring cell arrest in G2 phase, Cdc2A exhibited upregulation, favoring S phase progression. The impact of this mode of cell cycle regulation on viral infection remains unknown. However, cell arrest in G2 phase was described in other viral infections including human immunodeficiency virus (HIV) and was found to be mainly mediated by viral protein R (Vpr) [
91‐
93]. While many of the G2/M DNA damage checkpoint pathway genes are known to be modulated during HIV infection, Vpr seems to induce cell arrest by molecular mechanisms other than the classic DNA check point [
94]. Recently, evidence supporting a role for PP2A in Vpr-induced arrest has emerged, and was substantiated further by other studies in support of PP2A being a common target during infection with other viruses, including simian virus 40 (SV40), polyoma virus, human T lymphotrophic retrovirus and adenovirus [
95]. Our results showed significant downregulation in two PP2A isoforms, regulatory subunit B' gamma isoform (PPP2R5C) and protein phosphatase 2, regulatory subunit B' epsilon isoform (PPP2R5E), suggesting that the induction mechanisms of G2 arrest in MPV infection might be similar to those observed in other viruses. Because genetically diverse viruses seem to induce the same G2 arrest response in different infected cells, it is likely that this response has an important function and might be part of antiviral host defenses. While some of the viral genes eliciting this response are being identified as Vpr in HIV, and E4 of F4 and HTLV tax protein in adenoviruses, the MPV gene inducing this response remains unknown. Other important genes in cell cycle regulation showing expression favoring progression of cell cycle and arrest only in G2 phase include Rb, E2F, cyclins, BTG1, and BRCA1.
In this study we combined microarray with data mining and statistical analysis to identify important interfaces of host-pathogen interaction. Our results aligned nicely with previous reports carried out using viruses from the same or different genus, and provided new set of genes that play important roles in MPV infection. Further work is warranted to validate and examine the potential of these genes in antiviral therapies.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AA was responsible for design, conduct, and completion of this work, as well as for data analysis and writing of this manuscript. RH and MJ were instrumental in data processing and statistical analysis of microarray data. JH and MA contributed to microarray data validation using RT PCR. SI was the Principal Investigator and is primarily responsible for all aspects of the funding, research design, interpretation, and writing of this manuscript. All authors read and approved the final manuscript.