Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity

The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.     

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.     

Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death

Background  

Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310–380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.     

Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [125I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [125I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.     

Design of a UV-C irradiation process for the inactivation of viruses in protein solutions.

The ability of ultraviolet (UV) light to inactivate viruses is well established. However, attempts to apply this to the manufacture of pharmaceutical proteins have been limited by incomplete treatment, low capacity or excessive dilution. Effective processing of large-scale batches of UV-opaque protein solutions has been achieved using a continuous-flow device. The operation of this device has been modelled and a design equation derived to relate the processing conditions and product characteristics to the degree of virus inactivation obtained. Variables included in the model are UV-absorbance at 254 nm (A(254)), hydrodynamic properties of the protein solution, residence time, intensity of UV light and diameter and length of irradiation tube. With this information a specific constant was calculated for each virus which denotes its relative sensitivity to UV and from which the degree of virus inactivation expected can be estimated.     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.     

Photocatalytic inactivation of influenza virus by titanium dioxide thin film

Titanium dioxide (TiO(2)) under ultraviolet (UV) light produces a strong oxidative effect and may therefore be used as a photocatalytic disinfectant. Although many studies on the photocatalytic inactivation of bacteria have been reported, few studies have addressed virus inactivation. In the present study, we demonstrated the inactivation of influenza virus through TiO(2) photocatalysis using TiO(2) nanoparticles immobilized on a glass plate. The influences of the UV intensity, UV irradiation time and bovine serum albumin (BSA) concentration in the viral suspensions on the inactivation kinetics were investigated. Additionally, we also determined whether the International Organization for Standardization (ISO) methodology for the evaluation of antibacterial activity of TiO(2) photocatalysis could be applied to the evaluation of antiviral activity. The viral titers were dramatically reduced by the photocatalytic reaction. Even with a low intensity of UV-A (0.01 mW cm(-2)), a viral reduction of approximately 4-log(10) was observed within a short irradiation time. The viral inactivation kinetics were associated with the exposure time, the UV intensity and the BSA concentration in virus suspensions. These results show that TiO(2) photocatalysis could be used to inactivate the influenza virus. Furthermore, a minor modification of the ISO test method for anti-bacterial effects of TiO(2) photocatalysis could be useful for the evaluation of antiviral activity.     

Ultraviolet Inactivation Dichroic Ratio of Oriented fd Bacteriophage

Polarized UV light irradiation of flow-oriented fd bacteriophage indicates that the degree of damage (inactivation) depends on the relative orientation of the light polarization vector and the plane of the DNA bases. The technique of anisotropic UV inactivation was evaluated, and further information on the orientation in this virus was gained. The fd bacteriophage were aligned and irradiated with plane-polarized monochromatic UV light either parallel or perpendicular to the virus axis. Variation of the inactivation dichroic ratio with wavelength implicated virus inactivation by light absorbed in both the DNA and protein. Analysis of the wavelength variation of inactivation dichroic ratios gave molecular dichroic ratios of 0.76 and 1.48 for the DNA and protein components, respectively. On the basis of these anisotropic inactivation studies, the average angle of DNA base tilt in fd was calculated to be 29-32°, a value in agreement with the absorption dichroism studies of Bendet and Mayfield.     

Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus

Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is approximately 11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.     

DNA repair in cultured human fibroblasts does not decline with donor age

Human fibroblasts from young (3 days to 3 years) and old (84–94 years) donors were tested for their ability to repair DNA damage by measuring survival of colony formation following irradiation with ultraviolet (UV) light. Repair was also measured by the ability to reactivate herpes simplex virus following treatment of the virus with UV light, methyl methane sulfonate or 4,5′,8-trimethylpsoralen plus light. This virus was used as a probe of cellular repair capacity because survival of damaged virus is lower in repair-deficient cell lines [1]. Cell lines from both age groups exhibited comparable survivals following UV irradiation and failed to show increased sensitivity to irradiation in the presence of caffeine. Cells from both groups repaired damaged virus to equal extents. Proficient viral repair was observed under conditions in which cells were infected by either single or multiple viral genomes. These results suggest that DNA repair mechanisms which act on a variety of lesions (e.g. pyrimidine dimers, apurinic sites, alkylated bases, cross-links, etc.) do not decline with age. A model for biological aging resulting from the accumulation with age of unrepaired DNA damage is discussed.     

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Enhancement of UV-induced cytotoxicity by the adeno-associated virus replication proteins

Adeno-associated virus (AAV) normally requires co-infection of a helper virus to complete its life cycle. However, under conditions of cellular stress, such as treatment with carcinogens or ultraviolet (UV) light, a permissive intracellular environment is established and AAV completes its replicative cycle producing low levels of progeny virus. AAV DNA replication is dependent upon viral replication proteins, Rep78 and Rep68. The detailed mechanism by which these proteins interact with host cell factors is unknown. We have used a cell line (Neo6) that inducibly expresses the AAV Rep proteins to study their effects on cells that have undergone UV-induced DNA damage. Induction of Rep protein expression immediately after a sub-lethal dose of UV irradiation resulted in rapid cell killing. Those cells that die had chromatin condensation while cellular membranes remained intact, suggesting that concurrent Rep expression and UV damage induces an apoptosis-like response. However, we did not observe any DNA degradation. Thus we believe that the combination of Rep expression and UV irradiation induces cell death that shares some of the characteristics of apoptosis. UV irradiation and Rep expression induced an increase in the level of the CDK inhibitor, p21Cip, and the appearance of modified forms of both p21Cip and Bcl-2. Alteration of normal expression of these cytostatic/apoptotic proteins provides insight into the intracellular targets of the AAV replication proteins.     

Mechanisms involved in the protection of UV-induced protein inactivation by the corneal crystallin ALDH3A1

Various lines of evidence have shown that ALDH3A1 (aldehyde dehydrogenase 3A1) plays a critical and multifaceted role in protecting the cornea from UV-induced oxidative stress. ALDH3A1 is a corneal crystallin, which is defined as a protein recruited into the cornea for structural purposes without losing its primary function (i.e. metabolism). Although the primary role of ALDH3A1 in the metabolism of toxic aldehydes has been clearly demonstrated, including the detoxification of aldehydes produced during UV-induced lipid peroxidation, the structural role of ALDH3A1 in the cornea remains elusive. We therefore examined the potential contribution of ALDH3A1 in maintaining the optical integrity of the cornea by suppressing the aggregation and/or inactivation of other proteins through chaperone-like activity and other protective mechanisms. We found that ALDH3A1 underwent a structural transition near physiological temperatures to form a partially unfolded conformation that is suggestive of chaperone activity. Although this structural transition alone did not correlate with any protection, ALDH3A1 substantially reduced the inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal and malondialdehyde when co-incubated with NADP(+), reinforcing the importance of the metabolic function of this corneal enzyme in the detoxification of toxic aldehydes. A large excess of ALDH3A1 also protected glucose-6-phosphate dehydrogenase from inactivation because of direct exposure to UVB light, which suggests that ALDH3A1 may shield other proteins from damaging UV rays. Collectively, these data demonstrate that ALDH3A1 can reduce protein inactivation and/or aggregation not only by detoxification of reactive aldehydes but also by directly absorbing UV energy. This study provides for the first time mechanistic evidence supporting the structural role of the corneal crystallin ALDH3A1 as a UV-absorbing constituent of the cornea.     

Interferon Induction in Rabbit Cells Irradiated with UV Light

UV irradiation of a continuous line of rabbit kidney cells (RK13) was used as a tool for the study of the mechanism of interferon induction. Irradiation of cells prior to their exposure to Newcastle disease virus (NDV) resulted in a dose-dependent decrease in interferon production. The inhibition of total cellular RNA synthesis by UV irradiation in uninduced cultures was similar to the inactivation curve of interferon production in NDV-induced cultures. In contrast, the production of interferon with polyinosinate-polycytidylate (poly[I].poly [C]) paradoxically was enhanced in cells irradiated with a wide range of doses of UV. However, in cells stimulated with poly(I).poly(C) and "superinduced" by the sequential addition of cycloheximide and actinomycin D, the rate of inactivation of interferon production by UV light was similar to that observed with NDV. These results are not inconsistent with the idea that both poly(I).poly(C) and NDV stimulate the same interferon gene(s), but indicate that the mechanism controlling its expression may be different for each inducer.     

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.     

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.     

Early signaling events induced by 280-nm UV irradiation.

The depletion of stratospheric ozone results in increased UV (ultraviolet) light below 300 nm, and has significant effects on biological systems. To better understand the effects of UV in this range, early signaling events induced by monochromatic UV light were investigated using the chicken B cell line DT40 and mutants lacking protein tyrosine kinases (PTKs). Among MAP kinase family proteins, P38 MAP kinase (P38) was selectively and immediately activated by 280 nm UV light in cultured DT40 cells. Activation of P38 was completely inhibited in cells deficient in Lyn and Btk. Introduction of wild-type Btk, but not kinase-inactive Btk, restored the P38 activation. In contrast, P38 activation was not affected in Syk-deficient cells. Tyrosine phosphorylation of Lyn was induced by 280 nm UV light, and pretreatment of cells with orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP), enhanced both Lyn phosphorylation and P38 activation. These results show that Lyn and Btk are upstream regulators of the P38 signaling pathway activated by 280 nm UV light and that the triggering event likely involves inactivation of PTP. Furthermore, cell death induced by 280 nm UV irradiation were augmented by Btk depletion or a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that the Lyn-Btk-P38 pathway promotes cell survival while other Lyn pathways stimulate cell death.     

Generation and characterization of closely related epizootic and enzootic infectious cDNA clones for studying interferon sensitivity and emergence mechanisms of Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV) is a reemerging pathogen and a continuing threat to humans and equines in the Americas. Identification of the genetic determinants that enable epizootic VEEV strains to arise and exploit equines as amplification hosts to cause widespread human disease is pivotal to understanding VEE emergence. The sensitivity to murine alpha/beta interferon-mediated antiviral activity was previously correlated to the epizootic phenotype of several VEEV strains. Infectious cDNA clones were generated from an epizootic subtype IC VEEV strain (SH3) isolated during the 1992 Venezuelan outbreak and a closely related enzootic, sympatric subtype ID strain (ZPC738). These VEEV strains had low-cell-culture-passage histories and differed by only 12 amino acids in the nonstructural and structural proteins. Rescued viruses showed similar growth kinetics to their parent viruses in several cell lines, and murine infections resulted in comparable viremia and disease. Unlike what was found in other studies of epizootic and enzootic VEEV strains, the sensitivities to murine alpha/beta interferon did not differ appreciably between these epizootic versus enzootic strains, calling into question the reliability of interferon sensitivity as a marker of epizootic potential.