Genetic Analysis of the Emerging Citrus Yellow Vein Clearing Virus Reveals a Divergent Virus Population in American Isolates.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Unraveling the occasional occurrence of berry astringency in table grape cv. Scarlet Royal: a physiological and transcriptomic analysis.

Scarlet Royal, a mid-season ripening table grape, is one of the popular red grape varieties in California. However, its berries develop an undesirable astringent taste under certain conditions. Among the various factors contributing to the degradation of berry attributes, the levels and compositions of polyphenols play a fundamental role in defining berry quality and sensory characteristics. To comprehend the underlying mechanism of astringency development, Scarlet Royal berries with non-astringent attributes at the V7 vineyard were compared to astringent ones at the V9 vineyard. Biochemical analysis revealed that the divergence in berry astringency stemmed from alterations in its polyphenol composition, particularly tannins, during the late ripening stage at the V9 vineyard. Furthermore, transcriptomic profiling of berries positively associated nineteen flavonoid/proanthocyanidins (PAs) structural genes with the accumulation of PAs in V9 berries. The identification of these genes holds significance for table grape genetic improvement programs. At a practical level, the correlation between the taste panel and tannin content revealed a threshold level of tannins causing an astringent taste at approximately 400 mg/L. Additionally, berry astringency at the V9 vineyard was linked to a lower number of clusters and yield during the two study seasons, 2016 and 2017. Furthermore, petiole nutrient analysis at bloom showed differences in nutrient levels between the two vineyards, including higher levels of nitrogen and potassium in V9 vines compared to V7. It's worth noting that V9 berries at harvest displayed a lower level of total soluble solids and higher titratable acidity compared to V7 berries. In conclusion, our results indicate that the accumulation of tannins in berries during the ripening process results in a reduction in their red color intensity but significantly increases the astringency taste, thereby degrading the berry quality attributes. This study also highlights the association of high nitrogen nutrient levels and a lower crop load with berry astringency in table grapes, paving the way for further research in this area.

Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.

Transcriptome Analysis of Citrus Dwarfing Viroid Induced Dwarfing Phenotype of Sweet Orange on Trifoliate Orange Rootstock.

Dwarfed citrus trees for high-density plantings or mechanized production systems will be key for future sustainable citrus production. Citrus trees consist of two different species of scion and rootstock. Therefore, any observed phenotype results from gene expression in both species. Dwarfed sweet orange trees on trifoliate rootstock have been produced using citrus dwarfing viroid (CDVd). We performed RNA-seq transcriptome analysis of CDVd-infected stems and roots and compared them to non-infected controls. The identified differentially expressed genes validated with RT-qPCR corresponded to various physiological and developmental processes that could be associated with the dwarfing phenotype. For example, the transcription factors MYB13 and MADS-box, which regulate meristem functions and activate stress responses, were upregulated in the stems. Conversely, a calcium-dependent lipid-binding protein that regulates membrane transporters was downregulated in the roots. Most transcriptome reprogramming occurred in the scion rather than in the rootstock; this agrees with previous observations of CDVd affecting the growth of sweet orange stems while not affecting the trifoliate rootstock. Furthermore, the lack of alterations in the pathogen defense transcriptome supports the term "Transmissible small nuclear ribonucleic acid," which describes CDVd as a modifying agent of tree performance with desirable agronomic traits rather than a disease-causing pathogen.

QuantiGene Plex Assay: A Method for High-Throughput Multiplex Citrus Viroid Detection and Identification.

The QuantiGene Plex assay is a molecular non-polymerase chain reaction (PCR)-based multiplex method adapted for citrus viroid detection and identification. Here, we describe the procedures to utilize the QuantiGene Plex assay as a high-throughput screening tool for viroids in purified or crude RNA extracts from citrus tissues.

High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids.

High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.

SYBR® Green RT-qPCR for the Universal Detection of Citrus Viroids.

Quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR have now become the gold standard for molecular diagnostics because of its sensitivity, specificity, and reproducibility. In addition, qPCR diagnostics are flexible because they can be scaled for high- or low-throughput applications. Here we describe an optimized assay and workflow for the universal detection of eight citrus viroid species and their variants by RT-qPCR. The assay allows for quick and efficient molecular detection of viroids without the need to run RT-qPCR for each individual viroid species.

Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings.

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

Complete Nucleotide Sequence, Genome Organization, and Comparative Genomic Analyses of Citrus Yellow-Vein Associated Virus (CYVaV).

Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a -1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.

Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients.

BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.

Microsatellite characterization and marker development for the fungus Penicillium digitatum, causal agent of green mold of citrus.

Penicillium digitatum is one of the most important postharvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations. The genome of P. digitatum has been sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, a total of 3,134 microsatellite loci were detected; 66.73%, 23.23%, 8.23%, 1.24%, 0.16%, and 0.77% were detected as mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. As consistent with other ascomycete fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono-, di-, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and five additional isolates from other countries ("global isolates") were used to initially screen microsatellite markers developed in this study. Twelve additional isolates, referred to as the "local isolates," were also collected from citrus at the University of California Riverside agricultural experiment station and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty-six primers were screened, and nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded two to seven alleles and will be useful to study population genetic structure of P. digitatum populations.

Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.

Colorectal papillomavirus infection in patients with colorectal cancer.

PURPOSE: Infection with human papillomaviruses (HPV) is associated with the development of cervical cancer, but whether HPVs have a role in colorectal cancer remains controversial. EXPERIMENTAL DESIGNS: To determine the relationship between HPV and colorectal cancer, we did a retrospective, controlled study using tumor and tumor-adjacent colorectal tissues dissected from patients with colorectal cancer, as well as colorectal tissues from control individuals with no cancer. The samples were processed in a blinded fashion for nested PCR and in situ PCR detection of HPV DNAs. The PCR products were gel-purified and sequenced for HPV genotyping. RESULTS: We found that colorectal tissues from 28 of 55 (51%) patients with colorectal cancer were positive for HPV DNA. Colorectal tissues from all 10 control individuals were negative for HPV DNA (P = 0.0034). Of the 107 usable (GAPDH(+)) samples collected as paired colorectal tissues (tumor and tumor-adjacent tissues) from the patients, 38 (36%) had HPV16 (n = 31), HPV18 (n = 5), or HPV45 (n = 2), with HPV DNA in both tumor and tumor-adjacent tissues of 10 paired samples, 13 in only the tumor, and 5 in only tumor-adjacent tissues. In situ PCR detection of the tumor tissues confirmed the presence of HPV DNA in tumor cells. CONCLUSION: Our results suggest that colorectal HPV infection is common in patients with colorectal cancer, albeit at a low DNA copy number, with HPV16 being the most prevalent type. HPV infection may play a role in colorectal carcinogenesis.

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap.

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.

Natural Incidence of Mixed Infections and Experimental Cross Protection Between Two Genotypes of Tobacco mild green mosaic virus.

ABSTRACT Isolates of Tobacco mild green mosaic virus (TMGMV), a member of the genus Tobamovirus, from Nicotiana glauca in southern California fall into two major genotypes, large (TMGMV-L) and small (TMGMV-S), distinguishable by the size of the coat protein (CP) subgenomic RNA. Mixed infections in the field were rare (1.6%), even at sites where both genotypes were common in single infections (62% for TMGMV-S; 37% for TMGMV-L). When plants experimentally protected by TMGMV-L were challenged by TMGMV-S, almost complete cross protection (90% of total plants challenged) was observed regardless of the protective time period (minimum 12 h and maximum 14 days). When plants protected by TMGMV-S were challenged with TMGMV-L, complete cross protection was observed when the protective time was 5 to 14 days. However, when the protective time was 3 days or less, protection by TMGMV-S was greatly reduced (11%), with mixed infections of TMGMV-S and -L predominating (69%), and single infections of the challenge virus TMGMVL were frequently observed (20%). When TMGMV-S and -L virions were co-inoculated, the virus progeny from individual plants most often contained only the TMGMV-L genome (61%) or, less frequently (39%), both genotypes. Therefore, TMGMV-L was more competitive than TMGMV-S and was able to displace TMGMV-S in experimental situations. The results obtained from cross-protection experiments in the greenhouse would explain the low frequency of natural mixed infections. It is possible that the experimental superior competitiveness of the novel L genotype has already or will play a role in its abundance in southern California.

TUSC1, a putative tumor suppressor gene, reduces tumor cell growth in vitro and tumor growth in vivo.

We previously reported the identification of TUSC1 (Tumor Suppressor Candidate 1), as a novel intronless gene isolated from a region of homozygous deletion at D9S126 on chromosome 9p in human lung cancer. In this study, we examine the differential expression of TUSC1 in human lung cancer cell lines by western blot and in a primary human lung cancer tissue microarray by immunohistochemical analysis. We also tested the functional activities and mechanisms of TUSC1 as a tumor suppressor gene through growth suppression in vitro and in vivo. The results showed no expression of TUSC1 in TUSC1 homozygously deleted cells and diminished expression in some tumor cell lines without TUSC1 deletion. Interestingly, the results from a primary human lung cancer tissue microarray suggested that higher expression of TUSC1 was correlated with increased survival times for lung cancer patients. Our data demonstrated that growth curves of tumor cell lines transfected with TUSC1 grew slower in vitro than those transfected with the empty vector. More importantly, xenograph tumors in nude mice grew significantly slower in vivo in cells stably transfected with TUSC1 than those transfected with empty vector. In addition, results from confocal microscopy and immunohistochemical analyses show distribution of TUSC1 in the cytoplasm and nucleus in tumor cell lines and in normal and tumor cells in the lung cancer tissue microarray. Taken together, our results support TUSC1 has tumor suppressor activity as a candidate tumor suppressor gene located on chromosome 9p.

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites.

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.