UVC disinfects SARS-CoV-2 by induction of viral genome damage without apparent effects on viral morphology and proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a pandemic threat worldwide and causes severe health and economic burdens. Contaminated environments, such as personal items and room surfaces, are considered to have virus transmission potential. Ultraviolet C (UVC) light has demonstrated germicidal ability and removes environmental contamination. UVC has inactivated SARS-CoV-2; however, the underlying mechanisms are not clear. It was confirmed here that UVC 253.7 nm, with a dose of 500 µW/cm2, completely inactivated SARS-CoV-2 in a time-dependent manner and reduced virus infectivity by 10-4.9-fold within 30 s. Immunoblotting analysis for viral spike and nucleocapsid proteins showed that UVC treatment did not damage viral proteins. The viral particle morphology remained intact even when the virus completely lost infectivity after UVC irradiation, as observed by transmission electronic microscopy. In contrast, UVC irradiation-induced genome damage was identified using the newly developed long reverse-transcription quantitative-polymerase chain reaction (RT-qPCR) assay, but not conventional RT-qPCR. The six developed long RT-PCR assays that covered the full-length viral genome clearly indicated a negative correlation between virus infectivity and UVC irradiation-induced genome damage (R2 ranging from 0.75 to 0.96). Altogether, these results provide evidence that UVC inactivates SARS-CoV-2 through the induction of viral genome damage.

Monte Carlo Simulation of SARS-CoV-2 Radiation-Induced Inactivation for Vaccine Development.

Immunization with an inactivated virus is one of the strategies currently being tested towards developing a SARS-CoV-2 vaccine. One of the methods used to inactivate viruses is exposure to high doses of ionizing radiation to damage their nucleic acids. While gamma (γ) rays effectively induce lesions in the RNA, envelope proteins are also highly damaged in the process. This in turn may alter their antigenic properties, affecting their capacity to induce an adaptive immune response able to confer effective protection. Here, we modeled the effect of sparsely and densely ionizing radiation on SARS-CoV-2 using the Monte Carlo toolkit Geant4-DNA. With a realistic 3D target virus model, we calculated the expected number of lesions in the spike and membrane proteins, as well as in the viral RNA. Our findings showed that γ rays produced significant spike protein damage, but densely ionizing charged particles induced less membrane damage for the same level of RNA lesions, because a single ion traversal through the nuclear envelope was sufficient to inactivate the virus. We propose that accelerated charged particles produce inactivated viruses with little structural damage to envelope proteins, thereby representing a new and effective tool for developing vaccines against SARS-CoV-2 and other enveloped viruses.

Effectiveness of 222-nm ultraviolet light on disinfecting SARS-CoV-2 surface contamination.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has emerged as a serious threat to human health worldwide. Efficient disinfection of surfaces contaminated with SARS-CoV-2 may help prevent its spread. This study aimed to investigate the in vitro efficacy of 222-nm far-ultraviolet light (UVC) on the disinfection of SARS-CoV-2 surface contamination. METHODS: We investigated the titer of SARS-CoV-2 after UV irradiation (0.1 mW/cm2) at 222 nm for 10-300 seconds using the 50% tissue culture infectious dose (TCID50). In addition, we used quantitative reverse transcription polymerase chain reaction to quantify SARS-CoV-2 RNA under the same conditions. RESULTS: One and 3 mJ/cm2 of 222-nm UVC irradiation (0.1 mW/cm2 for 10 and 30 seconds) resulted in 88.5 and 99.7% reduction of viable SARS-CoV-2 based on the TCID50 assay, respectively. In contrast, the copy number of SARS-CoV-2 RNA did not change after UVC irradiation even after a 5-minute irradiation. CONCLUSIONS: This study shows the efficacy of 222-nm UVC irradiation against SARS-CoV-2 contamination in an in vitro experiment. Further evaluation of the safety and efficacy of 222-nm UVC irradiation in reducing the contamination of real-world surfaces and the potential transmission of SARS-CoV-2 is needed.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Ultraviolet germicidal irradiation of the inner bore of a CT gantry.

PURPOSE: To investigate the feasibility and practicality of ultraviolet (UV) germicidal irradiation of the inner bore of a computed tomography (CT) gantry as a means of viral decontamination. METHOD: A UV lamp (PADNUT 38 W, 253 nm UV-C light tube) and UV-C dosimeter (GENERAL UV-C Digital Light Meter No. UV512C) were used to measure irradiance throughout the inner bore of a CT scanner gantry. Irradiance (units µW/cm2 ) was related to the time required to achieve 6-log viral kill (10-6 survival fraction). RESULTS: A warm-up time of ~120 s was required for the lamp to reach stable irradiance. Irradiance at the scan plane (z = 0 cm) of the CT scanner was 580.9 µW/cm2 , reducing to ~350 µW/cm2 at z = ±20 cm toward the front or back of the gantry. The angular distribution of irradiation was uniform within 10% coefficient of variation. A conservative estimate suggests at least 6-log kill (survival fraction ≤ 10-6 ) of viral RNA within ±20 cm of the scan plane with an irradiation time of 120 s from cold start. More conservatively, running the lamp for 180 s (3 min) or 300 s (5 min) from cold start is estimated to yield survival fraction <<10-7 survival fraction within ±20 cm of the scan plane. CONCLUSION: Ultraviolet irradiation of the inner bore of the CT gantry can be achieved with a simple UV-C lamp attached to the CT couch. Such practice could augment manual wipe-down procedures, improve safety for CT technologists or housekeeping staff, and could potentially reduce turnover time between scanning sessions.

Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay.

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Radio-Susceptibility of Nasopharyngeal Carcinoma: Focus on Epstein- Barr Virus, MicroRNAs, Long Non-Coding RNAs and Circular RNAs.

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer. As a neoplastic disorder, NPC is a highly malignant squamous cell carcinoma that is derived from the nasopharyngeal epithelium. NPC is radiosensitive; radiotherapy or radiotherapy combining with chemotherapy are the main treatment strategies. However, both modalities are usually accompanied by complications and acquired resistance to radiotherapy is a significant impediment to effective NPC therapy. Therefore, there is an urgent need to discover effective radio-sensitization and radio-resistance biomarkers for NPC. Recent studies have shown that Epstein-Barr virus (EBV)-encoded products, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), which share several common signaling pathways, can function in radio-related NPC cells or tissues. Understanding these interconnected regulatory networks will reveal the details of NPC radiation sensitivity and resistance. In this review, we discuss and summarize the specific molecular mechanisms of NPC radio-sensitization and radio-resistance, focusing on EBV-encoded products, miRNAs, lncRNAs and circRNAs. This will provide a foundation for the discovery of more accurate, effective and specific markers related to NPC radiotherapy. EBVencoded products, miRNAs, lncRNAs and circRNAs have emerged as crucial molecules mediating the radio-susceptibility of NPC. This understanding will improve the clinical application of markers and inform the development of novel therapeutics for NPC.

Inactivation of Bombyx mori macula-like virus under physical conditions.

The Bombyx mori macula-like virus (BmMLV) is a member of the genus Maculavirus, family Tymoviridae, and contains a positive-sense single-stranded RNA genome. Previously, we reported that almost all B. mori-derived cell lines have already been contaminated with BmMLV via an unknown infection route. Since B. mori-derived cell lines are used for the baculovirus expression vector system, the invasion of BmMLV will cause a serious safety risk in the production of recombinant proteins. In this study, to determine the inactivation effectiveness of BmMLV, viruses were treated with various temperatures as well as gamma and ultraviolet (UV) light radiation. After these treatments, the virus solutions were inoculated into BmMLV-free BmVF cells. At 7 days postinoculation, the amount of virus in cells was evaluated by real-time reverse transcription PCR. Regarding heat treatment, conditions under 56°C for 3 h were tolerated, whereas infectivity disappeared after treatment at 75°C for 1 h. Regarding gamma radiation treatment, viruses were relatively stable at 1 kGy; however, their infectivity was entirely eliminated at a dose of 10 kGy. With 254 nm UV-C treatment, viruses were still active at less than 120 mJ/cm(2); however, their infectivity was completely lost at greater than 140 mJ/cm(2) UV-C radiation. These results provide quantitative evidence of the potential for BmMLV inactivation under a variety of physical conditions.

Comparison of UV-Induced Inactivation and RNA Damage in MS2 Phage across the Germicidal UV Spectrum.

Polychromatic UV irradiation is a common method of pathogen inactivation in the water treatment industry. To improve its disinfection efficacy, more information on the mechanisms of UV inactivation on microorganisms at wavelengths throughout the germicidal UV spectrum, particularly at below 240 nm, is necessary. This work examined UV inactivation of bacteriophage MS2, a common surrogate for enteric pathogens, as a function of wavelength. The bacteriophage was exposed to monochromatic UV irradiation from a tunable laser at wavelengths of between 210 nm and 290 nm. To evaluate the mechanisms of UV inactivation throughout this wavelength range, RT-qPCR (reverse transcription-quantitative PCR) was performed to measure genomic damage for comparison with genomic damage at 253.7 nm. The results indicate that the rates of RNA damage closely mirror the loss of viral infectivity across the germicidal UV spectrum. This demonstrates that genomic damage is the dominant cause of MS2 inactivation from exposure to germicidal UV irradiation. These findings contrast those for adenovirus, for which MS2 is used as a viral surrogate for validating polychromatic UV reactors.

The effectiveness of riboflavin photochemical-mediated virus inactivation and changes in protein retention in fresh-frozen plasma treated using a flow-based treatment device.

BACKGROUND: A flow-based treatment device using riboflavin and ultraviolet (UV) light was developed to inactivate viruses in fresh-frozen plasma (FFP). The objective of this study was to evaluate the in vitro effectiveness of virus inactivation and changes in protein quality in FFP treated with this device. STUDY DESIGN AND METHODS: FFP-contaminating viruses were treated with riboflavin and UV light using a one-pass linear flow device. The infectivity of viruses was measured using established biologic assays. Real-time polymerase chain reaction (PCR) was performed to detect damage to viral nucleotides after treatment. Treated plasma was analyzed using standard coagulation assays. RESULTS: FFP treated at the UV dose of 3.6 J/cm(2) (J) exhibited a mean reduction of virus titer of more than 4 logs. The effectiveness increased significantly at higher doses. Real-time PCR showed that the cycle threshold values for both complete inactivation and virus recultivation were higher than that of the untreated sample. At doses of 3.6, 5.4, and 7.2 J, the protein recovery rates were 60.2 ± 8.6, 46.6 ± 9.4, and 28.0 ± 1.0% for fibrinogen; 67.0 ± 3.1, 57.3 ± 8.0, and 49.2 ± 3.8% for Factor VIII; 93.6 ± 2.8, 89.6 ± 6.1, and 86.5 ± 5.3% for antithrombin-III; and 72.1 ± 5.6, 59.8 ± 14.2, and 49.2 ± 8.4% for Protein C, respectively. CONCLUSION: The effectiveness of virus inactivation was enhanced, but total activity of plasma factors was reduced, in a UV dose-dependent manner.

Capsid-Damaging Effects of UV Irradiation as Measured by Quantitative PCR Coupled with Ethidium Monoazide Treatment.

The damage to a viral capsid after low-pressure (LP) and medium-pressure (MP) UV irradiation was assessed, using the quantitative or quantitative reverse transcription PCR coupled with ethidium monoazide treatment (EMA-PCR). After UV irradiation, adenovirus 5 (Ad5) and poliovirus 1 (PV1) were subjected to a plaque assay, PCR, and EMA-PCR to investigate the effect of UV irradiation on viral infectivity, genome damage, and capsid damage, respectively. The effectiveness of UV wavelengths in a viral genome and capsid damage of both PV1 and Ad5 was also further investigated using a band-pass filter. It was found that an MPUV lamp was more effective than an LPUV lamp in inactivating Ad5, whereas there was no difference in the case of PV1. The results of viral reduction determined by PCR and EMA-PCR indicated that MP UV irradiation damaged Ad5 capsid. The damage to PV1 and Ad5 capsid was also not observed after LP UV irradiation. The investigation of effects of UV wavelengths suggested that UV wavelengths at 230-245 nm have greater effects on adenovirus capsid in addition to viral genome than UV wavelengths beyond 245 nm.

UVC Inactivation of dsDNA and ssRNA Viruses in Water: UV Fluences and a qPCR-Based Approach to Evaluate Decay on Viral Infectivity.

Disinfection by low-pressure monochromatic ultraviolet (UVC) radiation (253.7 nm) became an important technique to sanitize drinking water and also wastewater in tertiary treatments. In order to prevent the transmission of waterborne viral diseases, the analysis of the disinfection kinetics and the quantification of infectious viral pathogens and indicators are highly relevant and need to be addressed. The families Adenoviridae and Polyomaviridae comprise human and animal pathogenic viruses that have been also proposed as indicators of fecal contamination in water and as Microbial Source Tracking tools. While it has been previously suggested that dsDNA viruses may be highly resistant to UVC radiation compared to other viruses or bacteria, no information is available on the stability of polyomavirus toward UV irradiation. Here, the inactivation of dsDNA (HAdV2 and JCPyV) and ssRNA (MS2 bacteriophage) viruses was analyzed at increasing UVC fluences. A minor decay of 2-logs was achieved for both infectious JC polyomaviruses (JCPyV) and human adenoviruses 2 (HAdV2) exposed to a UVC fluence of 1,400 J/m(2), while a decay of 4-log was observed for MS2 bacteriophages (ssRNA). The present study reveals the high UVC resistance of dsDNA viruses, and the UV fluences needed to efficiently inactivate JCPyV and HAdV2 are predicted. Furthermore, we show that in conjunction with appropriate mathematical models, qPCR data may be used to accurately estimate virus infectivity.

Bacteriophage inactivation by UV-A illuminated fullerenes: role of nanoparticle-virus association and biological targets.

Inactivation rates of the MS2 bacteriophage and (1)O(2) generation rates by four different photosensitized aqueous fullerene suspensions were in the same order: aqu-nC(60) < C(60)(OH)(6) ≈ C(60)(OH)(24) < C(60)(NH(2))(6). Alterations to capsid protein secondary structures and protein oxidation were inferred by detecting changes in infrared vibrational frequencies and carbonyl groups respectively. MS2 inactivation appears to be the result of loss of capsid structural integrity (localized deformation) and the reduced ability to eject genomic RNA into its bacterial host. Evidence is also presented for possible capsid rupture in MS2 exposed to UV-A illuminated C(60)(NH(2))(6) through TEM imagery and detection of RNA infrared fingerprints in ATR-FTIR spectra. Fullerene-virus mixtures were also directly visualized in the aqueous phase using a novel enhanced darkfield transmission optical microscope fitted with a hyperspectral imaging (HSI) spectrometer. Perturbations in intermolecular extended chains, HSI, and electrostatic interactions suggest that inactivation is a function of the relative proximity between nanoparticles and viruses and (1)O(2) generation rate. MS2 log survival ratios were linearly related to CT (product of (1)O(2) concentration C and exposure time T) demonstrating the applicability of classical Chick-Watson kinetics for all fullerenes employed in this study. Results suggest that antiviral properties of fullerenes can be increased by adjusting the type of surface functionalization and extent of cage derivatization thereby increasing the (1)O(2) generation rate and facilitating closer association with biological targets.

RNA self-cleavage activated by ultraviolet light-induced oxidation.

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C(79) and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U(78) and CSFV U(44) in others. Among other reaction products, the majority of biochemically active products detected contained 3'-phosphate and 5'-phosphate-end groups at the newly generated termini, along with a much lower amount of 3'-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (~33 nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures.

Effect of radiation therapy to immunological and virological status in HIV/AIDS-cancer patients, preliminary report.

OBJECTIVE: To describe effects of radiation therapy (RT) on immunological status (CD4 cell counts) and disease progression among HIV-positive cancer patients. MATERIAL AND METHOD: This prospective observational study was conducted among HIV-positive cancer patients who received RT for curative intention of cancer in five selected hospitals in Thailand. All subjects received external beam radiation therapy, according to standard clinical practice guidelines of RT. Blood samples were taken 4 times for complete blood count, CD4 cell count and plasma HIV RNA viral load (HIV-VL) assays before and in the last week of RT, then three and six months after completion of RT. RESULTS: This preliminary study reported immunological status and HIV-VL before and the last week of RT, among 29 HIV-positive female cancer patients enrolled from August 22, 2009 to June 30, 2010. The median age was 38 years (range 30-54). 27 patients (93 percent) had invasive cervical cancer. 26 patients (90 percent) were on antiretroviral treatment (ART). The mean baseline white blood cell (WBC) count, lymphocyte percentage were 6,771.7 cells/microL and 31.7 percent respectively. The mean baseline CD4 cell count and CD4%, 387.8 cells/microL and 17.5 percent respectively. In the last week of RT, 25 subjects (86 percent) had CD4 count less than 200 cells/microL. The last week, mean WBC count, and mean lymphocyte percentage decreased to 3,902.8 cells/microL and 17.5 percent respectively. Mean CD4 count number decreased to 157.7 cells/microL, but the mean CD4 % did not change. Four patients (14 percent) had increased HIV-VL after RT, of these two were not on ART and two were on ART for more than 1 year. CONCLUSION: The CD4 cell count was not a good surrogate for prediction of immunologic status of HIV-positive cancer patients during RT.

[Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S ribosomal subunit].

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the outer subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome.

The 5'-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

Inactivation of viruses on surfaces by ultraviolet germicidal irradiation.

In many outbreaks caused by viruses, the transmission of the agents can occur through contaminated environmental surfaces. Because of the increasing incidence of viral infections, there is a need to evaluate novel engineering control methods for inactivation of viruses on surfaces. Ultraviolet germicidal irradiation (UVGI) is considered a promising method to inactivate viruses. This study evaluated UVGI effectiveness for viruses on the surface of gelatin-based medium in a UV exposure chamber. The effects of UV dose, viral nucleic acid type (single-stranded RNA, ssRNA; single-stranded DNA, ssDNA; double-stranded RNA, dsRNA; and double-stranded DNA, dsDNA), and relative humidity on the virus survival fraction were investigated. For 90% viral reduction, the UV dose was 1.32 to 3.20 mJ/cm2 for ssRNA, 2.50 to to 4.47 mJ/cm2 for ssDNA, 3.80 to 5.36 mJ/cm2 for dsRNA, and 7.70 to 8.13 mJ/cm2 for dsDNA. For all four tested viruses, the UV dose for 99% viral reduction was 2 times higher than those for 90% viral reduction. Viruses on a surface with single-stranded nucleic acid (ssRNA and ssDNA) were more susceptible to UV inactivation than viruses with double-stranded nucleic acid (dsRNA and dsDNA). For the same viral reduction, the UV dose at 85% relative humidity (RH) was higher than that at 55% RH. In summary, results showed that UVGI was an effective method for inactivation of viruses on surfaces.

Structural differences within the loop E motif imply alternative mechanisms of viroid processing.

Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multibranched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson-Crick GC pairs results in a significant overall stabilization of the CVd-III loop E-like motif. Unlike PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5' side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3' sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5' and 3' termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5' and 3' termini of CVd-III in position for the host ligase.

Evidence for the existence of the loop E motif of Potato spindle tuber viroid in vivo.

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.