Publications by authors named "Xibing Che"

16 Publications

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Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution.

Elife 2020 06 2;9. Epub 2020 Jun 2.

Division of Critical Care Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, United States.

At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.
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http://dx.doi.org/10.7554/eLife.56890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358008PMC
June 2020

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication.

Virology 2016 May 23;492:82-91. Epub 2016 Feb 23.

Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA, United States.

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.
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http://dx.doi.org/10.1016/j.virol.2016.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826839PMC
May 2016

ORF11 protein interacts with the ORF9 essential tegument protein in varicella-zoster virus infection.

J Virol 2013 May 20;87(9):5106-17. Epub 2013 Feb 20.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.
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http://dx.doi.org/10.1128/JVI.00102-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624291PMC
May 2013

Herpes simplex virus 1 tropism for human sensory ganglion neurons in the severe combined immunodeficiency mouse model of neuropathogenesis.

J Virol 2013 Mar 26;87(5):2791-802. Epub 2012 Dec 26.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

The tropism of herpes simplex virus (HSV-1) for human sensory neurons infected in vivo was examined using dorsal root ganglion (DRG) xenografts maintained in mice with severe combined immunodeficiency (SCID). In contrast to the HSV-1 lytic infectious cycle in vitro, replication of the HSV-1 F strain was restricted in human DRG neurons despite the absence of adaptive immune responses in SCID mice, allowing the establishment of neuronal latency. At 12 days after DRG inoculation, 26.2% of human neurons expressed HSV-1 protein and 13.1% expressed latency-associated transcripts (LAT). Some infected neurons showed cytopathic changes, but HSV-1, unlike varicella-zoster virus (VZV), only rarely infected satellite cells and did not induce fusion of neuronal and satellite cell plasma membranes. Cell-free enveloped HSV-1 virions were observed, indicating productive infection. A recombinant HSV-1-expressing luciferase exhibited less virulence than HSV-1 F in the SCID mouse host, enabling analysis of infection in human DRG xenografts for a 61-day interval. At 12 days after inoculation, 4.2% of neurons expressed HSV-1 proteins; frequencies increased to 32.1% at 33 days but declined to 20.8% by 61 days. Frequencies of LAT-positive neurons were 1.2% at 12 days and increased to 40.2% at 33 days. LAT expression remained at 37% at 61 days, in contrast to the decline in neurons expressing viral proteins. These observations show that the progression of HSV-1 infection is highly restricted in human DRG, and HSV-1 genome silencing occurs in human neurons infected in vivo as a consequence of virus-host cell interactions and does not require adaptive immune control.
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http://dx.doi.org/10.1128/JVI.01375-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571385PMC
March 2013

Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.

Proc Natl Acad Sci U S A 2012 Jan 21;109(2):600-5. Epub 2011 Dec 21.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.
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http://dx.doi.org/10.1073/pnas.1114232109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258638PMC
January 2012

Identification and functional characterization of the Varicella zoster virus ORF11 gene product.

Virology 2011 Mar 26;412(1):156-66. Epub 2011 Jan 26.

Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.
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http://dx.doi.org/10.1016/j.virol.2010.12.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068617PMC
March 2011

Varicella-zoster virus immediate-early protein 62 blocks interferon regulatory factor 3 (IRF3) phosphorylation at key serine residues: a novel mechanism of IRF3 inhibition among herpesviruses.

J Virol 2010 Sep 14;84(18):9240-53. Epub 2010 Jul 14.

Department of Pediatrics, Stanford University School of Medicine, Room S356, Grant Building, 300 Pasteur Drive, Stanford, CA 94305-5208, USA.

Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-beta production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-beta secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.
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http://dx.doi.org/10.1128/JVI.01147-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937611PMC
September 2010

Varicella-zoster virus T cell tropism and the pathogenesis of skin infection.

Curr Top Microbiol Immunol 2010 ;342:189-209

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.

Varicella-zoster virus (VZV) is a medically important human alphaherpesvirus that causes varicella and zoster. VZV initiates primary infection by inoculation of the respiratory mucosa. In the course of primary infection, VZV establishes a life-long persistence in sensory ganglia; VZV reactivation from latency may result in zoster in healthy and immunocompromised patients. The VZV genome has at least 70 known or predicted open reading frames (ORFs), but understanding how these gene products function in virulence is difficult because VZV is a highly human-specific pathogen. We have addressed this obstacle by investigating VZV infection of human tissue xenografts in the severe combined immunodeficiency mouse model. In studies relevant to the pathogenesis of primary VZV infection, we have examined VZV infection of human T cell (thymus/liver) and skin xenografts. This work supports a new paradigm for VZV pathogenesis in which VZV T cell tropism provides a mechanism for delivering the virus to skin. We have also shown that VZV-infected T cells transfer VZV to neurons in sensory ganglia. The construction of infectious VZV recombinants that have deletions or targeted mutations of viral genes or their promoters and the evaluation of VZV mutants in T cell and skin xenografts has revealed determinants of VZV virulence that are important for T cell and skin tropism in vivo.
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http://dx.doi.org/10.1007/82_2010_29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077053PMC
November 2010

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection.

Proc Natl Acad Sci U S A 2010 Jan 4;107(1):282-7. Epub 2009 Dec 4.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-DeltaP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-DeltaY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.
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http://dx.doi.org/10.1073/pnas.0912373107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806775PMC
January 2010

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection.

J Virol 2008 Jun 9;82(12):5825-34. Epub 2008 Apr 9.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.
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http://dx.doi.org/10.1128/JVI.00303-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395146PMC
June 2008

The ubiquitous cellular transcriptional factor USF targets the varicella-zoster virus open reading frame 10 promoter and determines virulence in human skin xenografts in SCIDhu mice in vivo.

J Virol 2007 Apr 24;81(7):3229-39. Epub 2007 Jan 24.

Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5208, USA.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) is a determinant of virulence in SCIDhu skin xenografts but not in human T cells in vivo. In this analysis of the regulation of ORF10 transcription, we have identified four ORF10-related transcripts, including a major 1.3-kb RNA spanning ORF10 only and three other read-through transcripts. Rapid-amplification-of-cDNA-ends experiments indicated that the 1.3-kb transcript of ORF10 has single initiation and termination sites. In transient expression assays, the ORF10 promoter was strongly stimulated by the major VZV transactivator, IE62. Deletion analyses revealed approximate boundaries for the full ORF10 promoter activity between -75 and -45 and between +5 and -8, relative to the ORF10 transcription start site. The recombinant virus POKA10-Deltapro, with the ORF10 promoter deletion, blocked transcription of ORF10 and also of ORF9A and ORF9 mRNAs, whereas expression of read-through ORF9A/9/10 and ORF9/10 transcripts was increased, compensating for the loss of the monocistronic mRNAs. The cellular factor USF bound specifically to its consensus site within the ORF10 promoter and was required for IE62 transactivation, whereas disrupting the predicted TATA boxes or Oct-1 binding elements had no effect. The USF binding site was disrupted in the recombinant virus, POKA10-proDeltaUSF, and no ORF10 protein was produced. Both ORF10 promoter mutants reduced VZV replication in SCIDhu skin xenografts. These observations provided further evidence of the contribution of the ORF10 protein to VZV pathogenesis in skin and demonstrated that VZV depends upon the cellular transcriptional factor USF to support its virulence in human skin in vivo.
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http://dx.doi.org/10.1128/JVI.02537-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866059PMC
April 2007

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo.

J Virol 2006 Dec 13;80(23):11806-16. Epub 2006 Sep 13.

Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5208, USA.

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.
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http://dx.doi.org/10.1128/JVI.00466-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1642581PMC
December 2006

Varicella-zoster virus open reading frame 10 is a virulence determinant in skin cells but not in T cells in vivo.

J Virol 2006 Apr;80(7):3238-48

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.

The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKA delta10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKA delta10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKA delta10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.
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http://dx.doi.org/10.1128/JVI.80.7.3238-3248.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440391PMC
April 2006

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements.

Virology 2003 Aug;312(2):434-48

The S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, The Volcani Center, 50250 Bet Dagan, Israel.

Grapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an approximately 7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Neither 3'- nor 5'-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5'-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3' end of 5.5-kb 5'-terminal sgRNA and the 5' end of the 1.8-kb 3'-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5'- and 3'-terminal sgRNAs was controlled by sequences upstream of the 5'-terminus of each of ORFs 2-4. Detection of both plus and minus strands of the 5'- and 3'-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3'-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3'-terminal minus-strand RNA, a 5'-terminal plus-strand sgRNA, and the corresponding 5'-terminal minus-strand RNA.
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http://dx.doi.org/10.1016/s0042-6822(03)00239-3DOI Listing
August 2003

Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs.

Virology 2003 Jun;310(2):298-309

The S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.

The family Closteroviridae includes the genera Closterovirus and Ampelovirus with monopartite genomes and the genus Crinivirus with bipartite genomes. Plants infected with the Closterovirus, Citrus tristeza virus (CTV), often contain one or more populations of defective RNAs (dRNAs). Although most dRNAs are comparatively small (2-5 kb) consisting of the genomic RNA termini with large internal deletions, we recently characterized large dRNAs of approximately 12 kb that retained the open reading frames (ORFs) 1a plus 1b. These were self-replicating RNAs and appeared to be analogous to the genomic RNA 1 of the bipartite criniviruses. The present report describes the finding of an additional group of large dRNAs (LdRNAs) that retained all or most of the 10 3' ORFs and appeared to be analogous to genomic RNA 2 of criniviruses. Isolates associated with LdRNAs were found associated with double-recombinant dRNAs (DR-dRNAs) of various sizes (1.7 to 5.1 kb) that comprised the two termini and a noncontiguous internal sequence from ORF2. The genetic and epidemiological implications of the architectural identities of LdRNAs and DR dRNAs and their apparent analogy with the genomic RNA 2 of criniviruses are discussed.
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http://dx.doi.org/10.1016/s0042-6822(03)00127-2DOI Listing
June 2003

A novel class of large and infectious defective RNAs of Citrus tristeza virus.

Virology 2002 Jun;298(1):133-45

The S. Tolkowsky Laboratory, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.

Citrus tristeza virus (CTV)-infected plants contain one or more populations of defective RNAs (dRNAs), mostly with a size range of ca. 2.0 to 5.0 kb. Several CTV dRNAs have been characterized and found to consist mainly of the two termini of the genomic RNA, with extensive internal deletions. The present paper describes a new class of large ( approximately 12.0 kb) dRNAs from three different CTV isolates with two unusual features. First is their composition with intact replicase genes. These dRNAs contained a large 5' portion of the genomic RNA terminus, which apparently corresponded to the recently described 5' large single-stranded subgenomic RNA (sgRNA) of ORF1a+1b (Che et al., 2001). The 3' portion of the large dRNAs varied among the 10 different cDNA clones examined in this work. In 2 dRNAs this portion consisted of truncated ORF10 (p20), and in 5 dRNAs it contained truncated ORF11 (p23). Two dRNA molecules were found with a 3' portion that started in the exact 5' position of the intergenic region between the p20 and p23 ORFs. In one dRNA, this portion coincided with the full-length sgRNA corresponding to ORF10. The second unusual feature was their ability to be readily transmitted mechanically to citrus plants by stem slashing and also to Nicotiana benthamiana protoplasts. The possibility that these dRNAs may be encapsidated and be capable of self-replication is discussed.
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http://dx.doi.org/10.1006/viro.2002.1472DOI Listing
June 2002