A UV-LED module that is highly effective at inactivating human coronaviruses and HIV-1.

Ultraviolet (UV) light has previously been established as useful method of disinfection, with demonstrated efficacy to inactivate a broad range of microorganisms. The advent of ultraviolet light-emitting diodes provides advantages in ease of disinfection, in that there can be delivery of germicidal UV with the same light unit that delivers standard white light to illuminate a room. Herein we demonstrate the efficacy and feasibility of ultraviolet light-emitting diodes as a means of decontamination by inactivating two distinct virus models, human coronavirus 229E and human immunodeficiency virus. Importantly, the same dose of ultraviolet light that inactivated human viruses also elicited complete inactivation of ultraviolet-resistant bacterial spores (Bacillus pumilus), a gold standard for demonstrating ultraviolet-mediated disinfection. This work demonstrates that seconds of ultraviolet light-emitting diodes (UV-LED) exposure can inactivate viruses and bacteria, highlighting that UV-LED could be a useful and practical tool for broad sanitization of public spaces.

Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States.

The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Env-activating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0°C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptor-binding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0°C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sensitivity by chemical agents that affect the structural stability of proteins can be applied to increase the potency of topical microbicides and vaccine-elicited antibodies.

Reduction of bacteria and human immunodeficiency virus Type 1 infectivity of platelet suspension in plasma using xenon flash-pulse light in a bench-scale trial.

BACKGROUND: Current pathogen reduction systems for platelet concentrates (PCs) require addition of chemical compounds and/or reduction of plasma content in PCs. We have investigated a new method using xenon (Xe) flash-pulse light without additional compounds or plasma replacement. STUDY DESIGN AND METHODS: An aliquot of apheresis platelets (PLTs) in plasma inoculated with bacteria or human immunodeficiency virus Type 1 (HIV-1) was irradiated with Xe flash-pulse light (Xe flash phototreatment). Bacterial growth was monitored up to 6 days of storage, whereas HIV-1 infectivity was assayed just after treatment. Pairs of Xe flash-phototreated and untreated PCs were examined for PLT lesion during the storage period. RESULTS: Under the current conditions, a low titer (1.8 colony-forming units [CFUs]/mL) of Staphylococcus aureus did not proliferate during the 6-day storage period, but grew in some cases at high-titer (24.0 CFUs/mL) inoculation. HIV-1 infectivity was reduced by 1.8 log. PLT recovery of the treated PCs was lower than untreated ones. An increase of mean PLT volume and glucose consumption, together with a decrease of hypotonic shock response and pH, were enhanced by the treatment. CD62P- and PAC-1-positive PLTs increased after the treatment, indicating the induction of PLT activation. Among biologic response modifiers, soluble CD40 ligand was significantly increased in the treated PCs on Day 6. CONCLUSIONS: Xe flash phototreatment could prevent bacterial proliferation and reduce HIV-1 infectivity in 100% plasma PCs without any additional compounds, but enhanced PLT storage lesions. Further improvement is required to increase the potency of pathogen inactivation with reducing PLT damage.

Transmission of human immunodeficiency virus Type-1 by fresh-frozen plasma treated with methylene blue and light.

BACKGROUND: The risk of transfusion-transmitted infection (TTI) has been minimized by introduction of nucleic acid testing (NAT) and pathogen inactivation (PI). This case report describes transmission of human immunodeficiency virus Type 1 (HIV-1) to two recipients despite these measures. STUDY DESIGN AND METHODS: In March 2009 a possible TTI of HIV-1 was identified in a patient that had received pooled buffy coat platelet concentrate (BC-PLT) in November 2005. The subsequent lookback study found two more patients who had received methylene blue (MB)-treated fresh-frozen plasma (FFP) and red blood cells (RBCs) from the same donation. In November 2005 the donor had tested negative for both HIV antibodies and HIV-1 RNA by 44 minipool (44 MP) NAT. Repository samples of this donation and samples from the recipients were used for viral load (VL) and sequence analysis. RESULTS: HIV-1 RNA was detectable by individual donation (ID)-NAT in the repository sample from the 2005 window period donation and a VL of 135 copies/mL was measured. HIV-1 infection was confirmed in both recipients of both BC-PLT (65 mL of plasma) and MB-FFP (261 mL of plasma), but not in the patient that had received 4-week-old RBCs (20 mL of plasma). The sequence analysis revealed a close phylogenetic relationship between the virus strains isolated from the donor and recipients, compatible with TTI. CONCLUSIONS: Approximately 17,600 and 4400 virions in the MB-FFP and BC-PLT were infectious, but 1350 virions in the RBCs were not. ID-NAT would have prevented this transmission, but the combination of MP-NAT and MB-PI did not.

Recurrence of cervical intraepithelial lesions after thermo-coagulation in HIV-positive and HIV-negative Nigerian women.

BACKGROUND: The burden of cervical cancer remains huge globally, more so in sub-Saharan Africa. Effectiveness of screening, rates of recurrence following treatment and factors driving these in Africans have not been sufficiently studied. The purpose of this study therefore was to investigate factors associated with recurrence of cervical intraepithelial lesions following thermo-coagulation in HIV-positive and HIV-negative Nigerian women using Visual Inspection with Acetic Acid (VIA) or Lugol's Iodine (VILI) for diagnosis. METHODS: A retrospective cohort study was conducted, recruiting participants from the cervical cancer "see and treat" program of IHVN. Data from 6 sites collected over a 4-year period was used. Inclusion criteria were: age ≥18 years, baseline HIV status known, VIA or VILI positive and thermo-coagulation done. Logistic regression was performed to examine the proportion of women with recurrence and to examine factors associated with recurrence. RESULTS: Out of 177 women included in study, 67.8 % (120/177) were HIV-positive and 32.2 % (57/177) were HIV-negative. Recurrence occurred in 16.4 % (29/177) of participants; this was 18.3 % (22/120) in HIV-positive women compared to 12.3 % (7/57) in HIV-negative women but this difference was not statistically significant (p-value 0.31). Women aged ≥30 years were much less likely to develop recurrence, adjusted OR = 0.34 (95 % CI = 0.13, 0.92). Among HIV-positive women, CD4 count <200cells/mm(3) was associated with recurrence, adjusted OR = 5.47 (95 % CI = 1.24, 24.18). CONCLUSION: Recurrence of VIA or VILI positive lesions after thermo-coagulation occurs in a significant proportion of women. HIV-positive women with low CD4 counts are at increased risk of recurrent lesions and may be related to immunosuppression.

Transient heating of expressed breast milk up to 65°C inactivates HIV-1 in milk: a simple, rapid, and cost-effective method to prevent postnatal transmission.

The postnatal transmission of human immunodeficiency virus (HIV) from mothers to children occurs through breastfeeding. Although heat treatment of expressed breast milk is a promising approach to make breastfeeding safer, it is still not popular, mainly because the recommended procedures are difficult to follow, or time-consuming, or because mothers do not know which temperature is sufficient to inactivate HIV without destroying the nutritional elements of milk. To overcome these drawbacks, a simple and rapid method of heat treatment that a mother could perform with regular household materials applying her day-to-day art of cooking was examined. This structured experiment has demonstrated that both cell-free and cell-associated HIV type 1 (HIV-1) in expressed breast milk could be inactivated once the temperature of milk reached 65°C. Furthermore, a heating method as simple as heating the milk in a pan over a stove to 65°C inhibited HIV-1 transmission retaining milk's nutritional key elements, for example, total protein, IgG, IgA, and vitamin B(12) . This study has highlighted a simple, handy, and cost-effective method of heat treatment of expressed breast milk that mothers infected with HIV could apply easily and with more confidence.

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.

Comparison of various radioactive payloads for a human monoclonal antibody to glycoprotein 41 for elimination of HIV-infected cells.

BACKGROUND: cART has significantly improved the life expectancy of people living with HIV (PLWH). However, it fails to eliminate the long-lived reservoir of latent HIV-infected cells. Radioimmunotherapy (RIT) relies on antigen-specific monoclonal antibodies (mAbs) for targeted delivery of lethal doses of ionizing radiation to cells. Previously, we have demonstrated that human mAb 2556 against HIV gp41 conjugated with 213Bismuth radioisotope (t1/2 = 46 min, alpha-emitter) selectively killed HIV-infected cells. 225Actinium (t1/2 = 9.92 d, alpha-emitter) and 177Lutetium (t1/2 = 6.7 d, beta-emitter) are two long-lived clinically proven radioisotopes for cancer treatment which might be more effective in killing infected cells systemically and in CNS. METHODS: In this study we have conjugated 2556 mAb with 213Bi, 225Ac and 177Lu, and compared their ability to kill HIV-infected human peripheral blood mononuclear cells (PBMCs) and monocytes. PBMCs and monocytes from healthy donors were infected with HIVp49.5 and treated in vitro with increasing concentrations of 213Bi (4-20 µCi)-, 225Ac (20-100 nCi)- and 177Lu (4-50 µCi)-2556 mAb. RESULTS: After three days post-treatment of infected PBMCs and monocytes, 213Bi- and 177Lu-conjugated 2556 mAb reduced virus production measured by p24 level in a dose-dependent manner, whereas, 225Ac-2556 showed minimal effect. However, seven days post-treatment all three radioisotopes showed significantly more pronounced reduction of virus replication as compared to control labeled mAb with 225Ac-2556 showing the least non-specific killing. CONCLUSION: These results indicate that RIT holds promise as a novel treatment option for the eradication of HIV-infected cells that merits further study in combination with cART and reactivation drugs.

Low-Level Ionizing Radiation Induces Selective Killing of HIV-1-Infected Cells with Reversal of Cytokine Induction Using mTOR Inhibitors.

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the "shock and kill" strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.

Comparative antiviral (HIV) photoactivity of metalized meso- tetraphenylsulfonated porphyrins.

We have carried out the study of the photochemical properties of a series of synthetic meso-tetraphenylsulfonated porphyrins (TPPMS4) bonded to several metal ions such as: Cu(II), Zn(II), Pd(II), Mn(II), Fe(III), Ni(II) and Co(II) for the optimization of their clinical applications as antiviral agents against the human immunodeficiency virus (HIV-1) as well as the study of the in vitro antiviral photoinactivation mechanisms with future application in blood sterilization. A selective inhibition has been determined in the viral growth (HIV-1) when this is irradiated in the presence of the complex TPPFeS4 and TPPMnS4 (photosensitizer-mediated Type I reaction) as well as in the 1O2-mediated (Type II reaction) in the presence of TPPPdS4 and TPPZnS4, remaining cellular viability unaltered in each case.

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein.

UV irradiation of human and murine cells enhances the transcription of several genes. Here we report on the primary target of relevant UV absorption, on pathways leading to gene activation, and on the elements receiving the UV-induced signal in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, in the gene coding for collagenase, and in the cellular oncogene fos. In order to induce the expression of genes. UV radiation needs to be absorbed by DNA and to cause DNA damage of the kind that cannot be repaired by cells from patients with xeroderma pigmentosum group A. UV-induced activation of the three genes is mediated by the major enhancer elements (located between nucleotide positions -105 and -79 of HIV-1, between positions -72 and -65 of the collagenase gene, and between positions -320 and -299 of fos). These elements share no apparent sequence motif and bind different trans-acting proteins; a member of the NF kappa B family binds to the HIV-1 enhancer, the heterodimer of Jun and Fos (AP-1) binds to the collagenase enhancer, and the serum response factors p67 and p62 bind to fos. DNA-binding activities of the factors recognizing the HIV-1 and collagenase enhancers are augmented in extracts from UV-treated cells. The increase in activity is due to posttranslational modification. While AP-1 resides in the nucleus and must be modulated there, NF kappa B is activated in the cytoplasm, indicating the existence of a cytoplasmic signal transduction pathway triggered by UV-induced DNA damage. In addition to activation, new synthesis of AP-1 is induced by UV radiation.

Loss of immune homeostasis dictates SHIV rebound after stem-cell transplantation.

The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4+ and CD8+ T cells and CD20+ B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev-induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells.

Inactivation of HCV and HIV by microwave: a novel approach for prevention of virus transmission among people who inject drugs.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) transmissions among people who inject drugs (PWID) continue to pose a challenging global health problem. Here, we aimed to analyse a universally applicable inactivation procedure, namely microwave irradiation, as a safe and effective method to reduce the risk of viral transmission. The exposure of HCV from different genotypes to microwave irradiation resulted in a significant reduction of viral infectivity. Furthermore, microwave irradiation reduced viral infectivity of HIV-1 and of HCV/HIV-1 suspensions indicating that this inactivation may be effective at preventing co-infections. To translate microwave irradiation as prevention method to used drug preparation equipment, we could further show that HCV as well as HIV-1 infectivity could be abrogated in syringes and filters. This study demonstrates the power of microwave irradiation for the reduction of viral transmission and establishment of this safety strategy could help reduce the transmission of blood-borne viruses.

Potential of Radiation-Induced Cellular Stress for Reactivation of Latent HIV-1 and Killing of Infected Cells.

The use of highly active antiretroviral therapy against HIV-1 for last two decades has reduced mortality of patients through extension of nonsymptomatic phase of infection. However, HIV-1 can be preserved in long-lived resting CD4(+) T cells, which form a viral reservoir in infected individuals, and potentially in macrophages and astrocytes. Reactivation of viral replication is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus (shock and kill strategy). In this opinion piece, we consider potential application of therapeutic doses of irradiation, the well-known and effective stress signal that induces DNA damage and activates cellular stress response, to resolve two problems: activate HIV-1 replication and virion production in persistent reservoirs under cART and deplete infected cells through selective cell killing using DNA damage responses.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.

Photodynamic inactivation of free and cell-associated HIV-1 using the photosensitizer, benzoporphyrin derivative.

The photosensitizer benzoporphyrin derivative monoacid ring A (BPD) has been investigated regarding its ability to destroy free and cell-associated human immunodeficiency virus type 1 (HIV-1) when activated by light. Experiments with free virus in tissue culture medium indicate that light-activated BPD was effective in rendering HIV uninfectious. Azidothymidine (AZT)-resistant strains of HIV appear equally susceptible to photodynamic inactivation under drug and light conditions that proved effective in inactivating AZT-sensitive strains of HIV. Experiments conducted on whole blood from individuals infected with HIV demonstrate that BPD and light treatment could significantly reduce cell-associated virus, under conditions that appear not to damage red blood cells. The amount of culturable virus from infected leukocytes surviving photodynamic treatment could be further reduced by the addition of AZT to the culture.

Activation of HIV type 1 long terminal repeat by ultraviolet light is serum and strain specific.

We have studied the UV responsiveness of xeroderma pigmentosum (XP) and HeLa cell lines transfected with a CAT reporter gene under the control of the HIV-1 LTR promoter. XP fibroblasts grown in 10% newborn bovine serum (NBS) were three times more responsive to UV radiation than cells grown in 10% fetal calf serum (FCS). Moreover, cocultivation of UV-irradiated XP cells with XP cells containing stable integrants of HIV-LTR CAT was found to be more than four times more effective in inducing the CAT activity when cells were maintained in 10% NBS than in 10% FCS. The level of induction was also dependent on the serum concentration. These data indicate that a serum component, possibly a cytokine(s), can enhance the UV response of both irradiated cells and unirradiated cells cocultivated with irradiated cells. The magnitude of UV responsiveness seemed also to be strain dependent. CAT activity for the HIV LTR promoter from the HTLV-IIIB (HIV-IIIB) strain was induced more than 30-fold by UV irradiation whereas activity from the LAV-1BRU strain was less than 2-fold. In contrast, both constructs were strongly induced by Tat expression. This indicates that there are differences in the induction mechanism for these two stimuli, even though UV radiation has been previously reported to induce a cellular Tat-like factor (Valerie K, et al., Nature [London] 1988;333:78-81).

Psoralen/UV inactivation of HIV-1-infected cells for use in cytologic and immunologic procedures.

A rapid procedure for the inactivation of HIV-1-infected cells using psoralen and ultraviolet (UV) light is described. Exposure of HIV-1-infected cells to 5 micrograms/ml psoralen followed by UV irradiation (320-380 nm) for 5 minutes yields cells that are noninfectious as assessed by extended infectivity assays. The psoralen/UV inactivation procedure described is effective with cells chronically or acutely infected with HIV-1 and is unaffected by cell densities up to 12 x 10(6)/ml. At 5 micrograms/ml psoralen does little damage to cellular permeability as shown by the ability of treated cells to exclude trypan blue and propidium iodide. Psoralen/UV treatment of HIV-1-infected cells does not cause a significant decrease in the reactivity of HIV-1 core and envelope antigens or cellular antigens to monoclonal antibodies. Experiments are presented demonstrating the use of these cells for flow cytometry studies and for cell surface labeling using the lactoperoxidase 125I iodination procedure.