Study on the Function of ORF Genes of Porcine Circovirus-like Virus P1

Libin WEN Xuejiao ZHU Qi XIAO Wei WANG Kongwang HE

Abstract ［Objectives］ This study was conducted to determine the functions of eight ORF genes of porcine circovirus-like virus P1.

［Methods］The double-copy tandem molecular cloning of porcine circovirus-like virus P1 genome was used to construct molecular clones with eight ORFs deleted by DNA site-directed mutagenesis technology. After transfected into PK15 cells for a certain period of time, RNA were extracted and was used to verify whether the eight ORFs were deleted or not and used for gene microarry analysis. The GO functions and KEGG pathway enrichment of differentially expressed genes were analyzed.

［Results］ P1 ORF1 is mainly involved in the biological processes of defense response to virus, signal transduction, regulation of Rab GTPase activity, and lipid metabolic process, and involved in the molecular functions of protein phosphatase inhibitor activity, phosphatidylinositol phospholipase C activity, 2 iron, 2 sulfur cluster binding, phosphoric diester hydrolase activity, and Rab GTPase activator activity, and in the KEGG pathways of secretion of digestive gland and nervous system development. P1 ORF2 is mainly involved in the biological processes of positive regulation of leukocyte chemotaxis, positive regulation of cell proliferation, positive regulation of cell migration, defense response to virus, regulation of cell growth, and involved in the molecular functions of insulin-like growth factor binding, and chemokine activity, and in the KEGG pathways of cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, toll-like receptor signaling pathway, chemokine signaling pathway, and cytokines, cytokine-cytokine receptor interaction. The biological processes, molecular functions and related pathways involving P1 ORF3 and ORF5 are basically similar to those of ORF2. P1 ORF8 is mainly involved in the biological processes of purine ribonucleotide biosynthetic process, amino acid transport, defense response to virus, amino acid transmembrane transport, and involved in molecular functions of N6-(1,2-dicarboxyethyl) AMP AMP-lyase (fumarate-forming) activity, iron-sulfur cluster binding, amino acid transmembrane transporter activity.

［Conclusions］The analysis of the ORF functions of P1 virus lays a foundation for the study of its pathogenicity and pathogenesis.

Key words Porcine circovirus-like virus P1; Function of ORF genes; Microarray; Differentially expressed genes

Received: November 4, 2020  Accepted: January 23, 2021

Supported by National Natural Science Foundation of China (30972184, 31272574).

Libin WEN (1967-), male, P. R. China, research follow, devoted to research about the prevention and control of animal diseases.

*Corresponding author. E-mail: wlbwhxjp@163.com.

Porcine circovirus is a member of Circovirus in Circoviridae, and four genotypes have been found. Among them, the porcine circovirus type 2 (PCV2) causes the porcine circovirus associated diseases (PCVADs), which have become important immunosuppressive diseases in pig production in China, especially the postweaning multisystemic wasting syndrome (PMWS), which is the most common clinically and more harmful. PMWS mainly occurs in piglets aged 5-12 weeks, and the clinical manifestations are progressive weight loss or growth retardation, anemia, and inguinal lymphadenopathy［1-6］. However, it is difficult to reproduce typical symptoms similar to clinical PMWS in animals infected with PCV2 alone. Some scholars believe that there is another "X" factor［7-8］. PCV2 has a variety of genotypes, of which the genome size is about 1 700 nucleotides. There are not only recombinations between different genotypes, but also defective viruses formed by genome rearrangement, and they may be porcine circovirus-like viruses that are formed through recombination with other molecules. Four types of porcine circovirus-like viruses have been found, temporarily named P1, P2, P3 and P4, respectively, of which the genome size is less than 1 000 nucleotides［9-13］. Therefore, the study of these porcine circovirus-like viruses will provide a new perspective for the study of PMWS, and it will also have practical and theoretical significance for the evolution of PCV2.

Among the four types of porcine circovirus-like viruses that have been discovered, the detection rate of porcine circovirus-like virus P1 is significantly higher than those of other three types of porcine circovirus-like viruses, so the research on virus P1 is more systematic. Virus P1 is a non-enveloped globular virus with a diameter of about 25 nm. It has a single-stranded circular DNA genome with a total length of 648 nucleotides. Except for 16 exogenous nucleotides, the remaining nucleotide sequences are highly homologous to the PCV2 capsid protein ORF2. It is the smallest known genome among animal viruses. The constructed double-copy tandem molecular clone of the P1 virus genome is infectious in vitro and in vivo, which is manifested by the occurrence of inclusion bodies in the cytoplasm and nucleus of the transfected PK15 cells, and the inoculated pigs have clinical symptoms similar to PMWS, such as progressive weight loss and anemia［14-16］. Although its genome is relatively short, the existence of 8 ORFs has been verified through PCR, RACE, Northern blot and other techniques. Among them, ORF1, ORF2, ORF4, and ORF5 are located on the negative strand of the P1 virus genome, at positions of 151-493, 492-579, 279-327, and 1-52, respectively, while ORF3, ORF6, ORF7, and ORF8 are located on the positive strand of the genome, at the positions of 494-599, 263-293, 285-336, and 374-419, respectively. It has been confirmed that ORF1 encodes the capsid protein of virus P1, which corresponds to part of the ORF2 nucleotide sequence of PCV2. P1 ORF1 lacks 1 nucleotide, so the C-terminus of the P1 capsid protein shares very low amino acid sequence homology with the protein encoded by ORF2 of PCV2［17］. In order to analyze the biological functions of the proteins or small peptides encoded by the 8 ORFs of virus P1, in this study, molecular clones that lack ORF1-ORF8 were constructed, respectively, using the previously constructed P1 infectious clones by DNA site-specific mutagenesis, and after transfecting cells with them for a certain period of time, DNA and RNA were harvested and extracted for use in virus replication and gene microarry analysis.

Materials and Methods

Cells and molecular clone of P1 virus genome

PK15 cells free from PCV1, PCV2, P1 and mycoplasma contamination were preserved by the Zoonotic Disease Prevention and Control Laboratory of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences. The double-copy tandem molecular clone of the porcine circovirus-like virus P1 genome was constructed and preserved by the Zoonotic Disease Prevention and Control Laboratory of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences［15-16］.

Main molecular biology reagents

Porcine Gene 1.1 ST Array Strip (901798) was a product of Affymetrix; the QuikChangek Multi Site-Directed Mutagenesis Kit was a product of Stratagene; TRIzol and LipofectamineTM 2000 liposome transfection reagents were products of Invitrogen; EndoFree Plasmid Kit was a product of QIAGEN; and the Mix (green) required for the PCR reaction was purchased from Beijing TSINGKE Xinye Biotechnology Co., Ltd.

Construction of molecular clones of P1 virus genome with deletion of one of ORF1-ORF8

The construction was conducted according to the instructions of the QuikChangek Multi Site-Directed Mutagenesis Kit. First, primers were designed for the deletion of ORF1-ORF8 in P1 molecular cloning, respectively, as shown in Table 1. The principle was to delete only specific ORF without introducing new ORFs, nor cause nonsense mutations and missenses in other overlapping ORFs.

Each P1 genome double-copy tandem molecular clone was used as a template, and PCR was performed using the primers in Table 1. The PCR started with pre-denaturation at 95 ℃ for 1 min, followed by a total of 30 cycles of 95 ℃ for 1 min, 55 ℃ for 1 min and 65 ℃ for 9 min. The product was cooled on ice for 2 min. Then, 1 μl of Dpn I enzyme was added to digest the parent plasmid free of mutation at 37 ℃ for 1 h. Next, XL 10-Gold super competent cells were transformed, and cultured with shaking in an NZY+ broth at 37 ℃ for 1 h. The culture was spread on a plate, and incubated at 37 ℃ for 16 h. Colonies were picked for sequencing to verify whether the mutation was successful.

Transfection

The PK-15 cells were transferred into a 6-well cell culture plate through cell passage, and when the cell density reached the condition that 90% or more cell fused, they were rinsed with serum-free OPTI-MEM I Medium. A certain amount of above plasmid DNA (4.0 μg) was diluted to 250 μl with OPTI-MEM I Medium, and a certain amount of LipofectamineTM 2000 (10 μl) was diluted to 250 μl. After standing at room temperature for 5 min, the two were mixed, and after standing at room temperature for 20 min, the mixture was added to a culture plate at 500 μl per well. After culturing in a CO2 incubator at 37 ℃ for 5 h, the medium was replaced with RPMI 1640 with 10% fetal calf serum to continue the culture. Meanwhile, untransfected PK-15 cells and PK-15 cells transfected with empty vector pSK were set as controls. Three replicates were set for each plasmid.

Gene microarry analysis of the biological functions of P1 ORF1-ORF8

At 12 h after the construction of the eight mutant P1 molecular clones, parental P1 molecular clones and PK15 cells transfected with empty vector pSK, they were harvested together with the cell control, and rinsed after discarding the culture medium. Then, TRIzol was added to extract total RNA according to the instructions, respectively, and the extracted total RNA was used for the detection of deleted gene transcripts and gene microarry analysis, the former of which was performed according to published literature［17］, and the latter was completed by Beijing CapitalBio Technology Co., Ltd. The basic process was as follows: each RNA sample was determined by the ultra-micro spectrophotometer NanoDrop for the concentration and purity of the sample, and double-stranded cDNA was synthesized from the purified total RNA, purified and transcribed in vitro into biotin-labeled cRNA, which was subjected to fragmentation, hybridization, eluation, staining, and microarry scanning to obtain differentially expressed miRNA. In this study, P<0.05 and fold change>1.2 were used to define differentially expressed genes (DEGs), and DEGs of interest were selected. Primers were designed and synthesized, and the DEGs displayed by the gene microarry were verified by real-time quantitative PCR. GO and KEGG function enrichment analysis were performed on the verified target genes, with Q<0.05 indicating that the differences were statistically significant.

Results and Analysis

Screening and verification of differentially expressed genes

Compared with the molecular clone of the female parent P1 genome, there were 12 differentially expressed genes with ORF1 deletion, of which 6 were up-regulated genes and 6 were down-regulated genes; there were 5 differentially expressed genes with ORF2 deletion, which were all down-regulated; with the deletion of ORF3, 1 gene was up-regulated and 4 genes were down-regulated; there were 3 differentially expressed genes with ORF4 deletion, which were all up-regulated; 4 differentially expressed genes with ORF5 deletion were all down-regulated; there was no change genes with ORF6 deletion; there was 1 gene with ORF7 deletion, which was up-regulated; and the deletion of ORF8 caused 3 genes to be up-regulated and 29 genes to be down-regulated (Table 2). From the genes with ORF1, ORF2, ORF3, ORF5 and ORF8 deletion, we selected IRG6 and TLR1, DDX60, IRG6 and CXCL10, CXCL10 and IRG6, CXXL10, DDX60 and IRG6, and ADSL, SLC7A2 and IRG6, respectively, 13 differentially expressed genes in total, which were verified by real-time quantitative PCR at the mRNA level. The expression changes of the selected genes were basically consistent with the results of the gene microarry (the specific process and related diagrams were omitted), indicating that the microarry results are reliable and can be used for subsequent analysis.

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GO function annotation of differentially expressed genes

GO has 3 ontology (ontology), mainly including biological process (BP), molecular function (MF) and cellular component CC), 3 parts in total. The GO function annotation results were given as below:

The differentially expressed genes caused by the deletion of ORF1 are mainly involved in biological processes of defense response to virus, signal transduction, regulation of Rab GTPase activity, and lipid metabolic process, involved in the molecular functions of protein phosphatase inhibitor activity, phosphatidylinositol phospholipase C activity, phosphoric diester hydrolase activity, and Rab-GTPase activator activity, and mainly located in lipid particles, endoplasmic reticulum membrane and other cellular components.

The differentially expressed genes caused by the deletion of ORF2 are mainly involved in the biological processes of leukocyte chemotaxis regulation, cell growth, proliferation and migration population, defense response to virus, involved in the molecular functions of insulin-like growth factor binding, and chemokine activity, and mainly located in lipid particles, endoplasmic reticulum, and extracellular components.

Similar to the deletion of ORF2, the differentially expressed genes caused by the deletion of ORF3 are mainly involved in biological processes of cell growth, proliferation and migration regulation, leukocyte chemotaxis regulation, and defense response to virus, mainly involved in the molecular functions of insulin-like growth factor binding, and chemokine activity, and mainly located in lipid particles, endoplasmic reticulum, and extracellular components. Similarly, the differentially expressed genes caused by the deletion of ORF5 are mainly involved in biological processes of cell proliferation and migration regulation, leukocyte chemotaxis regulation, defense response to virus, and immune response, mainly involved in molecular functions such as chemokine activity, and mainly located in lipid particles, endoplasmic reticulum and other cellular components.

The ifferentially expressed genes caused by the deletion of ORF8 are mainly involved in the biological processes of purine ribonucleotide biosynthetic process, amino acid transport and transmembrane trasport, and defense response to virus, mainly involved in the molecular functions  of N6-(1,2-dicarboxyethyl) AMP AMP-lyase (fumarate-forming) activity, iron-sulfur cluster binding, and amino acid transmembrane transporter activity, and mainly located in lipid particles, endoplasmic reticulum and other cellular components. The results are shown in Table 3.

KEGG pathway enrichment analysis of differentially expressed genes

The results of KEGG pathway enrichment analysis showed that pathways involving the differentially expressed genes caused by the deletion of ORF1 mainly include salivary secretion, gastric acid secretion, pancreatic secretion, endocrine and other factor-regulated calcium reabsorption, calcium signaling pathway, phosphoinositide metabolism, phosphatidylinositol signaling system, glutamatergic synapse, Alzheimers disease, Huntington disease, Wnt pathway, and GnRH signaling pathway; and main pathways involving the differentially expressed genes caused by the deletion of ORF2 are cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, toll-like receptor signaling pathway, chemokine signaling pathway, cytokines, and cytokine-cytokine receptor interaction. The same as those caused by the deletion of ORF2, the pathways in which the differentially expressed genes caused by the deletion of ORF3 and ORF5 are significantly expressed are also cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, toll-like receptor signaling pathway, chemokine signaling pathway, cytokines, and cytokine-cytokine receptor interaction. The results are shown in Table 4.

Discussion

So far, virus P1 is the DNA factor that infects animals with the smallest genome. It was first detected in the serum of pigs with clinical manifestations of PMWS, and later, it was showed that it can also infect cattle, sheep, rabbits and other animals, and is clinically closely related to PMWS, sow abortion, piglet congenital tremor, etc.［13,16,18-19］. Although the nucleotide sequence of the circular DNA genome of P1 is highly homologous to PCV2, and may be derived from the recombination of PCV2 with other molecules, P1 is not a defective virus of PCV2 and does not rely on PCV2 to infect animals. Although the genome is short, the detected P1 virus genome contains 8 ORFs. Therefore, the analysis of the functions of these ORFs is of great significance for understanding the characteristics of the virus, the pathogenic characteristics of the virus, and the interaction between the virus and the hosts. In this study, we used the P1 infectious clones constructed in the early stage to delete ORFs one by one through site-directed mutagenesis and transfect cells. The gene microarry technology was mainly employed to compare the clones with gene deletion with the parental clones on the basis of the changes in the host cell transcriptome, so as to analyze the biological functions of each ORF of P1.

An important part of gene microarry expression data analysis is to screen for differentially expressed genes, and most published papers have doubling the expression level or reducing the expression level by half as a criterion for judging whether there is a difference in expression, which is more consistent with the real-time quantitative PCR verification results, but it is also possible to miss genes that have minor changes in expression but are biologically important. The results of this study showed that after the deletion of most P1 ORFs, there were fewer differential genes whose expression levels were doubled or reduced by half compared with the parent, which might be related to the use of molecular clones instead of viruses, transfection efficiency, rescue virus protein expression level, selection of harvest time point and other factors. Referring to related literatures［20-21］, in this study, the expression level greater than 1.2 times was used as the criterion for screening differentially expressed genes, and after quantitative PCR verification, the expression results of randomly selected genes were basically the same as those of microarry hybridization.

The P1 virus ORF1 encodes 114 amino acids, which constitute the capsid protein of the virus. From the KEGG pathway enrichment analysis, it can be seen that P1 ORF1 is mainly involved in the secretion of digestive glands (saliva, gastric acid, pancreas), calcium reabsorption, etc., the changes of which directly affect the digestion and absorption of feed by animals, which might be the direct reason for the progressive weight loss of pigs infected with P1. In addition, P1 ORF1 is involved in the Wnt pathway that plays an important role in embryonic development, and certain neurodegenerative diseases, and piglet congenital tremor is closely related to P1. Whether it is related to ORF1 remains to be further studied, but follow-up studies have indeed confirmed that virus P1 can inhibit the Wnt pathway［22］.

The P1 virus ORF2, ORF3 and ORF5 encode 29, 35 and 17 amino acids, respectively, which are all small peptides in nature. From the KEGG pathway enrichment analysis, they are mainly involved in innate immunity and other pathways. In this study, the biological functions of P1 ORF4, ORF6, and ORF7 have not yet been resolved, which may be related to the biological activity of these small peptides, or may be caused by the harvest time. The P1 virus ORF8 encodes 15 amino acids, and its deletion caused the most differential gene expression. It is mainly involved in biological processes such as purine nucleotide biosynthesis, amino acid transport and transmembrane transport. However, in statistics applying Q value (P-value corrected value) analysis which is stricter than P value, the differentially expressed genes caused by the deletion of P1 ORF8 were not involved in any pathway.

Conclusions

The biological functions of the ORFs of the virus P1 genome were revealed using P1 infectious molecular cloning, mainly through gene microarry analysis and research. Among them, P1 ORF1 is mainly involved in digestive gland secretion and neurodevelopment, ORF2, ORF3 and ORF5 are mainly involved in innate immune response, etc., and ORF8 is mainly involved in biological processes such as purine nucleotide biosynthesis, amino acid transport and transmembrane transport.

References

［1］ TISCHER I, GELDERBLOM H, VETTERMANN W, et al. A very small porcine virus with circular single-stranded DNA［J］. Nature, 1982, 295(5844): 64-66.

［2］ ALLAN G, MEEHAN B, TODD D, et al. Novel porcine circovirus from pigs with wasting disease syndrome［J］. The Veterinary Record, 1998, 142(17): 467-468.

［3］ ELLIS J, HASSARD L, CLARK E, et al. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome［J］. The Canadian Veterinary Journal, 1998, 39(1): 44-51.

［4］ PALINSKI R, PI EYRO P, SHANG P, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure［J］. Journal of Virology, 2017, 91(1): e01879-16.

［5］ PHAN TG, GIANNITTI F, ROSSOW S, et al. Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation［J］. Virology Journal, 2016, 13(1): 184.

［6］ ZHANG HH, HU WQ, LI JY, et al. Novel Circovirus species identified in farmed pigs designated as porcine Circovirus 4, Hunan Province, China［J］. Transboundary and Emerging Diseases, 2020, 67(3): 1057-1061.

［7］ FENAUX M, HALBUR PG, HAQSHENAS G, et al. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions［J］. Journal of Virology, 2002, 76(2): 541-551.

［8］ ALLAN G, KRAKOWKA S, ELLIS J, et al. Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2［J］. Virus Research, 2012, 164(1-2): 4-9.

［9］ WEN L, WANG F, LI B, et al. Genetic diversity of intragenomic rearrangement of porcine circovirus type 2 in vitro and in vivo［J］. Agricultural Science & Technology, 2013, 14(12): 1719-1722.

［10］ WEN L, HE K, YU Z, et al. Complete genome sequence of a novel porcine circovirus-like agent［J］. Journal of Virology, 2012, 86(1): 639.

［11］ WEN LB, HE KW, YANG HC, et al. Complete nucleotide sequence of a novel porcine circovirus-like agent and its infectivity in vitro［J］. Science in China Series C: Life Sciences, 2008, 51(5): 1-6.

［12］ WEN L, WANG F, HE K, et al. Recombination in truncated genome sequences of porcine circovirus type 2［J］. Archives of Virology, 2015, 160(1): 371-374.

［13］ WEN L, MAO A, ZHU X, et al. Detection of a novel porcine circovirus-like agent in aborted pig foetuses［J］. Transboundary and Emerging Diseases, 2018, 65(5): 1364-1366.

［14］ WEN L, JIAO F, ZHANG D, et al. Molecular characterization of porcine circovirus-like virus P1 in eastern China［J］. Infection Genetics and Evolution, 2017(48): 54-57.

［15］ WEN L, HE K, YANG H. Infectivity analysis of cloned genomic DNA of P1 agent in vitro［J］. Chinese Journal of Animal and Veterinary Sciences, 2009(S1): 43-46.

［16］ WEN L, HE K, XIAO Q, et al. A novel porcine circovirus-like agent P1 is associated with wasting syndromes in pigs［J］. PLoS One, 2012, 7(8): e41565.

［17］ WEN L, WANG F, HE K, et al. Transcriptional analysis of porcine circovirus-like virus P1［J］. BMC Veterinary Research, 2014(10): 287.

［18］ WEN L, MAO A, FAN Z, et al. Porcine circovirus-like virus P1 in cattle, goats and rabbits in China［J］. Transboundary and Emerging Diseases, 2018, 65(1): e217-e218.

［19］ WEN L, MAO A, JIAO F, et al. Evidence of porcine circovirus-like virus P1 in piglets with an unusual congenital tremor［J］. Transboundary and Emerging Diseases, 2018, 65(2): e501-e504.

［20］ WANG Y, LIU C, FANG Y, et al. Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuis［J］. BMC Genomics, 2012(13): 68.

［21］ GUO A, CAI J, GONG W, et al. Transcriptome analysis in chicken cecal epithelia upon infection by eimeria tenella in vivo［J］. PLoS one, 2013, 8(5): e64236.

［22］ ZHU X, WEN L, SHENG S, et al. Porcine circovirus-like virus P1 inhibits Wnt signaling pathway in vivo and in vitro［J］. Frontiers in Microbiology, 2018(9): 390.

［23］ MANKERTZ A, HILLENBRANd B. Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene［J］. Virology, 2001, 279(2): 429-438.