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.

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.

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.

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.

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.

Predicted inactivation of viruses of relevance to biodefense by solar radiation.

UV radiation from the sun is the primary germicide in the environment. The goal of this study was to estimate inactivation of viruses by solar exposure. We reviewed published reports on 254-nm UV inactivation and tabulated the sensitivities of a wide variety of viruses, including those with double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA genomes. We calculated D(37) values (fluence producing on average one lethal hit per virion and reducing viable virus to 37%) from all available data. We defined "size-normalized sensitivity" (SnS) by multiplying UV(254) sensitivities (D(37) values) by the genome size, and SnS values were relatively constant for viruses with similar genetic composition. In addition, SnS values were similar for complete virions and their defective particles, even when the corresponding D(37) values were significantly different. We used SnS to estimate the UV(254) sensitivities of viruses for which the genome composition and size were known but no UV inactivation data were available, including smallpox virus, Ebola, Marburg, Crimean-Congo, Junin, and other hemorrhagic viruses, and Venezuelan equine encephalitis and other encephalitis viruses. We compiled available data on virus inactivation as a function of wavelength and calculated a composite action spectrum that allowed extrapolation from the 254-nm data to solar UV. We combined our estimates of virus sensitivity with solar measurements at different geographical locations to predict virus inactivation. Our predictions agreed with the available experimental data. This work should be a useful step to understanding and eventually predicting the survival of viruses after their release in the environment.

Viral activation of macrophages through TLR-dependent and -independent pathways.

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.

Human T-cell leukaemia virus type I is highly sensitive to UV-C light.

The biological characteristics of human T-cell leukaemia virus type I (HTLV-I) are not yet well understood. UV light C (UV-C) sensitivity of HTLV-I was studied using a newly established infectivity assay: infection with cell-free HTLV-I dose-dependently induced syncytial plaques in cat cells transduced with the tax1 gene of HTLV-I. HTLV-I was inactivated by a much lower UV dose than bovine leukaemia virus (BLV). The D(10) (10 % survival dose) of HTLV-I was about 20 J m(-2), while that of BLV was about 180 J m(-2), which was similar to the reported D(10) of BLV. The UV sensitivity of HTLV-I and BLV was also examined by detecting viral DNA synthesis 24 h after infection. The D(10) values determined by PCR using the gag primers for HTLV-I and BLV were close to those determined by the infectivity assays. Further PCR analyses were then performed to determine D(10) values using several different primers located between the 5'-long terminal repeat (5'-LTR) and the tax1 gene. The difference in UV sensitivity between HTLV-I and BLV was detected very early during replication, even during reverse transcription of the 5'-LTR of irradiated viruses, and became more prominent as reverse transcription proceeded towards the tax1 gene. Chimeric mouse retroviruses that contain the LTR-tax1 fragments of HTLV-I and BLV were made and hardly any difference in UV sensitivity was detected between them, suggesting that the difference was not determined by the linear RNA sequences of HTLV-I and BLV. HTLV-I was found to be much more sensitive than other retroviruses to UV.

Circulating Epstein-Barr virus DNA in serum of patients with lymphoepithelioma-like carcinoma of the lung: a potential surrogate marker for monitoring disease.

PURPOSE: The purpose of this work was to study the sera of patients with lymphoepithelioma-like carcinoma (LELC) of the lung for circulating EBV DNA. EXPERIMENTAL DESIGN: Prospectively collected serum samples from five female patients with advanced, inoperable LELC of the lung were measured for free circulating EBV DNA using a quantitative PCR technique. EBV-encoded small RNA (EBER)-1 was assayed in serial serum samples of three of the five patients, either from the start or during the initial phase of chemotherapy/radiotherapy until their terminal event or last follow-up. There was only a single-point sample for analysis in the fourth and fifth patients. Six other patients with LELC of the lung were also retrospectively identified, and their sera were tested for EBER-1 at either the first visit plus the last follow-up visit (n = 2), the first visit only (n = 2), or the last follow-up visit only (n = 2). RESULTS: Prospectively collected serum samples from five patients and retrospectively collected serum samples from two patients who had clinical disease at initial serum measurement showed detectable levels of EBER-1. Retrospectively collected serum samples from four patients with no clinical disease had negative sera. There is consistent correlation between the clinical response to treatment and subsequent clinical course of LELC and serum EBER-1 levels in the three prospective patients with longitudinal serum monitoring. CONCLUSIONS: This study shows for the first time that free EBV DNA can be detected in the serum of patients with LELC of the lung and further suggests the feasibility of its use for monitoring response to therapy in advanced cases.

Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.

Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation.

The UVC irradiation doses necessary for a 99.9% (3-log) inactivation of 3 different fish pathogenic viruses diluted in freshwater/seawater and wastewater from a fish processing plant were determined. The results showed that both infectious salmon anaemia virus (ISAV) and viral haemorrhagic septicaemia virus (VHSV) were very sensitive to UVC irradiation, showing a 3-log reduction of infectivity in freshwater of 33 +/- 3.5 and 7.9 +/- 1.5 J m(-2), respectively, while that of infectious pancreatic necrosis virus (IPNV) was substantially higher, 1188 +/- 57 J m(-2). Using ISAV as a model, a comparison of the effect of UVC irradiation on virus isolation versus reverse transcription polymerase chain reaction (RT-PCR) showed that considerably higher UVC doses, depending on the length of the amplified product, were necessary to abolish RT-PCR detection of viral RNA.

[Effect of microwave pretreatment on the detection of Epstein-Barr virus EBER RNA's using in situ hybridization]. / Effets du prétraitement aux micro-ondes sur la détection des ARN EBER du virus d'Esptein-Barr (EBV) par hybridation in situ.

We have tested the influence of microwave pretreatment of tissue sections on in situ hybridization applied to the detection of Epstein-Barr virus-encoded small RNAs (EBER). To this end, different protocols have been tested on slides of 4 lymph node lymphomas: microwave pretreatment, proteinase K pretreatment, microwave followed by proteinase K and no pretreatment. To compare the sensitivity of each technique, several dilutions of oligoprobes were used. Hybrids have been detected by immunohistochemistry. The analysis of the nuclear stainings obtained shows an enhancement of the sensitivity in protocols using microwaves.

Target structures for HIV-1 inactivation by methylene blue and light.

In a photodynamic virus inactivation procedure for human fresh frozen plasma the plasma is exposed to visible light in the presence of 1 microM methylene blue. This procedure is known to inactivate HIV-1 by at least 10(6.32) TCID50/ml within 10 minutes. To elucidate the mechanism of photodynamic inactivation of HIV-1 by methylene blue/light treatment, reverse transcriptase (RT), the HIV-1 associated protein p24, and viral RNA were examined. In the dark, methylene blue up to 10 microM has no inhibitory effect on recombinant RT. In the presence of light, recombinant RT inactivation was dependent on illumination time and the concentration of methylene blue. After photoinactivation of the whole virus by methylene blue/light treatment, RT activity was also almost completely inhibited. Simultaneously, it was found by Western blotting that HIV-1 p24 and gp120 are altered in size, possibly due to protein cross-linking. In addition, it was shown by polymerase chain reaction (PCR) inhibition assay that HIV-1 inactivation leads to destruction of its RNA. In summary, methylene blue/light treatment acts on HIV-1 at different target sites: the envelope and core proteins, and the inner core structures RNA and RT.

The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region.

Initiation of translation of poliovirus RNA by ribosomal entry into an internal segment of the 742-nucleotide (nt)-long 5' nontranslated region involves trans-acting factors, including p57, a 57-kDa polypeptide which has been identified as the pyrimidine tract-binding protein (PTB). A UV cross-linking assay was used to compare the RNA-binding properties of the p57 present in various mammalian cytoplasmic extracts with those of purified murine p57 and recombinant human PTB. Three noncontiguous p57-binding sites were located within the poliovirus 5' nontranslated region, between nt 70 and 288, and 443 and 539 (domain V), and 630 and 730. With the same assay, a novel 34-kDa polypeptide was identified that bound nt 1 to 629 specifically. A single A-->G substitution of nt 480 which attenuates poliovirus did not alter UV cross-linking of p57 to domain V. Although UV cross-linking of p57 to the internal ribosome entry site was specifically reduced by competition with poly(U) but not by competition with poly(C), poly(G), and poly(A) homoribopolymers, the presence of a polyuridine tract was not a sufficient determinant for binding of RNA to the p57 present in cytoplasmic extracts, nor was the polypyrimidine tract downstream of domain V necessary for binding to this site.