The sequence necessary for the infectivity of hop stunt viroid cDNA clones

Summary Recombinant plasmids carrying more than two units of the hop stunt viroid (HSV) cDNA sequence linked in tandem were highly infectious. On the other hand, those carrying one unit were not. To determine the length of the HSV cDNA sequence necessary for the infectivity, we constructed series of plasmids carrying more than one but less than two units of the HSV sequence and investigated their infectivities. The results showed that cDNA clones were infectious if about 60 nucleotides of the internal part of the HSV sequence (region A) were undeleted and remained with the same arrangement as in the infectious two-unit clone. The region A can be folded into a stable secondary structure. Viroid replication is thought to involve a site-specific endonucleolytic cleavage of plus strand RNA multimers produced by a rolling circle mechanism to yield unit length molecules. Our results suggest that the region A contains the sequence or structure recognized by the putative endonuclease.     

中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT—PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3’末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5a中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到10^7～10^8PFU／ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaⅠ酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。     

Feline calicivirus VP2 is essential for the production of infectious virions

The third open reading frame (ORF3) located at the 3′ end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5′ end and small deletions in the 3′ end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3′ end.     

Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.     

Reverse Genetics Identifies the Product of Open Reading Frame 4 as an Essential Particle Assembly Factor of Nyamanini Virus

We established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly.     

A Novel Strategy To Develop a Robust Infectious Hepatitis C Virus Cell Culture System Directly from a Clinical Isolate

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10⁵ focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.     

Contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection

Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.