Jiaqi Lu

Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus.

Abstract The overlapping genomic regions coding for structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) poses problems for molecular dissection of the virus replication process. We constructed five mutant full-length cDNA clones with the overlapping regions unwound and 1 to 3 restriction sites inserted between two adjacent ORFs (ORF1/2, ORF4/5, ORF5/6, ORF 6/7 and ORF7/3′ UTR), which generated the recombinant viruses. Our findings demonstrated that 1) the overlapping structural protein ORFs can be physically separated, and is dispensable for virus viability; 2) such ORF separations did not interrupt the subgenomic RNA synthesis; 3) the plaque morphology, growth kinetics, and antigenicity of these mutant viruses were virtually indistinguishable from those of the parental virus in cultured cells; and 4) these mutant viruses remained genetic stable in vitro. This study lays a foundation for further molecular dissection of PRRSV replication process, and development of genetically tagged vaccines against PRRS.

Recombinant PRRSV expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus.

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) expresses its genes via a set of nested subgenomic (sg) mRNAs. Such discontinuous transcription is unique yet poorly understood for arterivirus. The utilization of transcription-regulating sequence (TRS) remains a puzzle, as many TRS-like sequences exist in viral genome, yet only six or seven sg mRNAs were transcribed in arterivirus infected cells. To investigate the transcriptional control of the porcine arterivirus, a recombinant PRRSV infectious cDNA clone pCPV expressing the capsid gene of porcine circovirus 2 (PCV2) between PRRSV ORF1b and ORF2a was developed. The rescued recombinant viruses contained a range of disparate deletions of the inserted PCV2 sequence, yet two stable recombinant viruses containing 41 and 275nt of foreign sequences were generated upon plaque purification and serial passages. Further analysis of the sg RNA2 profile revealed that an array of novel sg RNA species was generated in cells infected with the recombinant virus. One group was formed by utilizing the inserted PCV2 sequence as TRS; another group was generated from cryptic TRS-like PRRSV sequences located 19, 37 and 97nt immediately downstream of the PRRSV ORF2 AUG. These results demonstrated that (1) the recombinant virus from direct insertion of foreign sequences was genetically unstable, while two recombinant PRRSVs containing foreign sequence of 41 or 275nt in length, respectively, became stable upon plaque purification and further serial passages; (2) PRRSV can utilize foreign TRS-like sequence as transcriptional promoter; (3) the insertion of foreign sequence provoked the generation of novel subgenomic RNAs utilizing cryptic TRS-like sequences that remain non-functional in native PRRSV.

Cis-acting structural element in 5′ UTR is essential for infectivity of porcine reproductive and respiratory syndrome virus

Abstract It is believed that the genomic 5′ untranslated region (UTR) of Arterivirus plays crucial roles in viral genomic replication, subgenomic mRNA transcription and protein translation, yet the structure and function still remain largely unknown. In this study, we conducted serial nucleotide truncation, ranging from 1 to 190 nucleotides, to the 5′ UTR of the porcine reproductive and respiratory syndrome virus (PRRSV) infectious full-length cDNA clone pAPRRS. In vitro synthetic RNAs were transfected into MARC-145 cells for further genetic and virologic analysis. Our results demonstrated that the first three nucleotides of PRRSV 5′ UTR were dispensable for virus viability, which however was repaired with foreign sequences. In order to assess if the primary sequence or structural element play more important regulatory roles, the CMV promoter-driven 5′ UTR truncation mutant cDNA clones were directly transfected into the BHK-21 cell lines. We found that PRRSV tolerated the first 16 nucleotides sequence alteration of 5′ UTR without losing virus viability. However, these revertant viruses contained a range of non-templated with unknown origin exogenous nucleotides in the repaired 5′ end. Further analyses revealed that the 5′ proximal stem–loop 1 (SL1) in the highly structured 5′ UTR was invariably required for virus infectivity. Taken together, we conclude that authentic 5′-proximal primary sequence is nonessential, but the resultant structural elements are probably indispensable for PRRSV infectivity.

Polymorphism of the PRNP gene in the main breeds of indigenous Chinese goats

The polymorphism of the PRNP gene plays a key role in susceptibility to prion disease. Scrapie is a neurodegenerative disease affecting sheep and goats and belongs to the group of prion diseases. We isolated DNA from 333 goat samples representing the main local goat breeds in six provinces in China to identify PRNP polymorphisms and to determine whether these breeds were at risk for developing scrapie. Two novel amino acid polymorphisms (R211G and T219I) and a novel silent mutation at codon 125 as well as nine previously reported polymorphisms were observed. Twenty-eight alleles and forty-nine different genotypes were obtained. The codon 142M associated with resistance of goat scrapie was not found in this study. The codon 143R was relatively rare. The codon 222K, a potentially useful candidate site for selecting for scrapie resistance, was also rare in indigenous Chinese goats. These results could provide some useful data for assessing the risk of scrapie in Chinese indigenous goats.

Replacement of the heterologous 5′ untranslated region allows preservation of the fully functional activities of type 2 porcine reproductive and respiratory syndrome virus

Abstract The 5′ untranslated region (UTR) is believed to be vital for the replication of porcine reproductive and respiratory syndrome virus (PRRSV), yet its functional mechanism remains largely unknown. In this study, to define the cis-acting elements for viral replication and infectivity, The 5′ UTR swapping chimeric clones pTLV8 and pSHSP5 were constructed based on two different genotypes full-length infectious cDNA clone pAPRRS and pSHE backbones. Between them, vTLV8 could be rescued from pTLV8 and had similar virological properties to vAPRRS, including phenotypic characteristic and RNA synthesis level. However, pSHSP5 exhibited no evidence of infectivity. Taken together, the results presented here demonstrate that only the 5′ UTR of type 1 PRRSV did not affect the infectivity and replication of type 2 PRRSV in vitro. The 5′ UTR of type 2 PRRSV could be functionally replaced by its counterpart from type 1.

Conserved nucleotides in the terminus of the 3′ UTR region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-15517AAUU15520-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-15503AACCA15507-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex.

Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity

The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.