HOCl Rapidly Kills Corona, Flu, and Herpes to Prevent Aerosol Spread.

The COVID-19 pandemic has escalated the risk of SARS-CoV-2 transmission in the dental practice, especially as droplet-aerosol particles are generated by high-speed instruments. This has heightened awareness of other orally transmitted viruses, including influenza and herpes simplex virus 1 (HSV1), which are capable of threatening life and impairing health. While current disinfection procedures commonly use surface wipe-downs to reduce viral transmission, they are not fully effective. Consequently, this provides the opportunity for a spectrum of emitted viruses to reside airborne for hours and upon surfaces for days. The objective of this study was to develop an experimental platform to identify a safe and effective virucide with the ability to rapidly destroy oral viruses transported within droplets and aerosols. Our test method employed mixing viruses and virucides in a fine-mist bottle atomizer to mimic the generation of oral droplet-aerosols. The results revealed that human betacoronavirus OC43 (related to SARS-CoV-2), human influenza virus (H1N1), and HSV1 from atomizer-produced droplet-aerosols were each fully destroyed by only 100 ppm of hypochlorous acid (HOCl) within 30 s, which was the shortest time point of exposure to the virucide. Importantly, 100 ppm HOCl introduced into the oral cavity is known to be safe for humans. In conclusion, this frontline approach establishes the potential of using 100 ppm HOCl in waterlines to continuously irrigate the oral cavity during dental procedures to expeditiously destroy harmful viruses transmitted within aerosols and droplets to protect practitioners, staff, and other patients.

Evidence for the involvement of a nuclear NF-kappa B inhibitor in global down-regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells.

Diminished expression of major histocompatibility complex class I antigens on the surface of adenovirus type 12 (Ad12)-transformed cells contributes to their high tumorigenic potential by enabling them to escape immune recognition by cytotoxic T lymphocytes. This low class I antigen expression is due to a block in class I transcription, which is mediated by Ad12 E1A. Genetic analysis has shown that the class I enhancer is the target for transcriptional down-regulation. In this study, we show that the ability of the R1 element of the class I enhancer to stimulate transcription is greatly reduced in Ad12-transformed cells. The loss of functional activity by the R1 element was attributed to loss of binding by the NF-kappa B p50-p65 heterodimer. NF-kappa B binding appears to be blocked within the nucleus rather than at the level of nuclear translocation. Significantly, NF-kappa B binding activity could be recovered from the nuclear extracts of Ad12-transformed cells following detergent treatment, suggesting that the block is mediated through a nuclear inhibitor present in the Ad12-transformed cells. These results, taken together with the fact that the R2 element of the class I enhancer exhibits strong binding to the transcriptional repressor COUP-TF, suggest that the class I enhancer is globally down-regulated in Ad12-transformed cells.

trans-dominant mutants of E1A provide genetic evidence that the zinc finger of the trans-activating domain binds a transcription factor.

The 289R E1A protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by E1A is mediated through cellular transcription factors. In particular, the ability of the trans-dominant E1A point mutant hr5 (Ser-185 to Asn) to inhibit wild-type E1A trans-activation was proposed to result from the sequestration of a cellular factor. Using site-directed mutagenesis, we individually replaced every residue within and flanking the trans-activating domain with a conservative amino acid, revealing 16 critical residues. Six of the individual substitutions lying in a contiguous stretch C terminal to the zinc finger (carboxyl region183-188) imparted a trans-dominant phenotype. trans-Dominance was even produced by deletion of the entire carboxyl region183-188. Conversely, an intact finger region147-177 was absolutely required for trans-dominance, since second-site substitution of every critical residue in this region abrogated the trans-dominant phenotype of the hr5 protein. These data indicate that the finger region147-177 bind a limiting cellular transcription factor and that the carboxyl region183-188 provides a separate and essential function. In addition, we show that four negatively charged residues within the trans-activating domain do not comprise a distinct acidic activating region. We present a model in which the trans-activating domain of E1A binds to two different cellular protein targets through the finger and carboxyl regions.

An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation.

The E1A gene of adenovirus type 5 encodes a 289-amino-acid (289R) protein that transactivates early adenovirus promoters. We showed that the 289R protein of the E1A missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) E1A protein from stimulating transcription from each of the early viral promoters E2, E3, and E4. Since both the hr5 and wt genes produced similar levels of E1A proteins, the ability of hr5 E1A to block transactivation was attributed to the replacement of serine by asparagine as position 185. We confirmed that this single amino acid substitution was responsible for blocking transactivation by showing equal inhibition with an hr5-wt hybrid E1A gene containing this missense mutation as the only alteration. The smaller 243R E1A protein of hr5 was not necessary for inhibition. Transcriptional activity from each early promoter was inhibited by at least 50% when the hr5 and wt E1A genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other E1A missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt E1A-induced transcription when similarly tested. Also, the hr5 E1A gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 E1A inhibits wt E1A activation at the transcriptional, rather than the posttranscriptional, level. Although several possibilities were considered to account for this inhibition, the most likely is that the nonfunctional hr5 E1A protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.

Reduced phosphorylation of p50 is responsible for diminished NF-kappaB binding to the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells.

Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-kappaB in adenovirus-transformed cells.

Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex.

Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex.

Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.

The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.

BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.

Human melanoma cells transcribe interleukin 1 genes identical to those of monocytes.

Two molecular species of the pleotropic cytokine interleukin 1 (IL-1) are produced as products of two distinct genes transcribed by cells of the monocyte-macrophage lineage. We have shown previously that a significant proportion of human melanoma cell lines express IL-1 biological activity, but it has not been demonstrated that this activity is the same as authentic monocyte IL-1 alpha and -beta. Here we report the cloning and sequencing of IL-1 complementary DNAs from a metastatic melanoma cell line and demonstrate that they encode bona fide IL-1 alpha and IL-1 beta. In addition, IL-1 complementary DNAs encoding a different amino acid at position 145 were revealed.

Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression.

The melanoma-associated antigen ME491 is expressed strongly during the early stages of tumor progression. The ME491 gene was molecularly cloned by means of DNA-mediated gene transfer followed by screening a lambda genomic library with human repetitive Alu sequences as a probe. The cloned DNA, after transfection into mouse L-cells, generated a protein with characteristics that were indistinguishable in Western blot analysis from the ME491 antigen expressed by human melanoma cells. Repeat-free subfragments of the cloned DNA were used for further studies. By Northern blot analysis, the subfragments detected a single 1.2-kilobase mRNA in the transformants and various human melanoma cell lines. ME491 complementary DNA clones were then obtained by probing a melanoma complementary DNA library with the genomic subfragments. Nucleotide sequence analysis of the cloned complementary DNA indicated that the ME491 antigen consists of 237 amino acids (Mr 25,475) with four transmembrane regions and three putative N-glycosylation sites. No significant structural homology was observed with other proteins thus far reported. We observed that the amounts of mRNA varied greatly with different melanoma cell lines. Southern blot analysis revealed no amplification or rearrangement of the ME491 gene in the human melanoma cell lines tested, including both high and low expressors of this antigen. The ME491 gene has been mapped to chromosome region 12p12----12q13 by somatic cell hybrid analysis and more narrowly localized to 12q12----12q14 by in situ hybridization.

Modulation of major histocompatibility complex (MHC) class I genes in adenovirus 12 transformed cells: interferon-gamma increases class I expression by a mechanism that circumvents E1A induced-repression and tumor necrosis factor enhances the effect of interferon-gamma.

The protein products of the E1A gene of adenovirus type-12 (Ad12) block transcription of major histocompatibility (MHC) class I genes in both rodent and human transformed cells and interferon-gamma (IFN-gamma) is able to override this repression. Although IFN-gamma is known to stimulate class I transcription, we investigated whether its dominance over E1A repression could alternatively result from the ability of this cytokine to induce antiviral mechanisms. We show that this is not so, since the accumulation of Ad12 E1A mRNA and protein are unabated in the presence of IFN-gamma. Also, tumor necrosis factor (TNF) was shown to act synergistically with IFN-gamma to enhance class I antigen levels, although it had little effect alone. These results suggest that the normal pathway by which IFN-gamma acts to enhance the level of class I mRNAs, circumvents the block by which E1A represses class I transcription.

Down-regulation of MHC class I antigen in insulinoma cells controlled by the R1 element of the H-2 enhancer.

Tumorigenesis in mice of the rat insulin promoter [RIP]-simian virus 40 tumor antigen [SV40 Tag] transgenic lineages, RIP1-Tag2 and RIP1-Tag4, is a process initiated by expression of SV40 Tag in pancreatic beta cells, evolution of islet cell hyperplasia and insulinoma appearance. Analysis of major histocompatibility complex [MHC] class I gene expression during this process revealed a normal level of MHC class I molecules at the surface of pancreatic islet cells of RIP1-Tag4 mice, while hyperplastic islets from the same mice contained cells expressing a normal level and cells expressing a low level of MHC class I antigen. Insulinomas themselves expressed very low levels or no MHC class I gene product. Thus, down-regulation of MHC class I gene appears to accompany tumor progression of SV40 Tag-transformed beta islet cells. MHC class I antigen expression in a series of clonally derived cell lines of beta cell origin from different SV40 Tag-induced insulinomas ranged from quite low to undetectable, although expression was inducible by interferon-gamma. Nuclear run-on and transient transfection analyses indicated that expression of the MHC class I gene in these cells in controlled at the transcriptional level, and that the decreased expression is paralleled by reduced binding of transcription factors to the R1 element of the H-2 enhancer.

Testing NF-kappa B1-p50 antibody specificity using knockout mice.

Cell extracts from knockout mice can provide definitive proof of antibody specificity. Two NF-kappa B1-p50 antibodies, sc-114 (a commercial antibody) and NR1157, were observed to recognize proteins having distinct electrophoretic mobilities of 52-55 kD and 50 kD, respectively, by Western blot analysis. In order to discriminate the specificity of these antibodies for NF-kappa B1-p50, whole cell extracts derived from NF-kappa B1-p105 knockout mice were employed. While the NR1157 antibody completely failed to recognize its 50 kD product in p105-/- knockout extracts, the sc-114 antibody still strongly recognized its 52-55 kD product. These data demonstrate that NR1157, but not sc-114, is highly specific for NF-kappa B1-p50 by Western blot analysis. In addition, these results highlight the utility of knockout cell extracts for discerning antibody specificity.

The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110.

A function shared by the adenovirus E1A, papillomavirus E7 and SV40 TAg oncoproteins is their ability to interfere with normal cell growth by interacting with members of the retinoblastoma protein family. In this study, we show that each of these oncoproteins can also bind to the 921 amino acid TBP-associated factor-110 (TAF-110). The significance of the binding is underscored by the observation that each oncoprotein binds to the same 77 amino acid carboxyl region of TAF-110. In the case of E1A and TAg, this finding is consistent with their abilities to stimulate transcription initiation, in part, through their known interactions with TBP. While it is not clear whether E7 can also activate promoters through protein:protein interactions with components of the transcription initiation complex, our demonstration that E7 can bind to TAF-110, as well as TBP, suggests that E7 may modulate the expression of specific promoters which could contribute to the pathogenesis of human papillomavirus.

The first exon of Ad12 E1A excluding the transactivation domain mediates differential binding of COUP-TF and NF-kappa B to the MHC class I enhancer in transformed cells.

The major histocompatibility complex class I enhancer is the target for adenovirus-12 E1A-mediated down-regulation of class I transcription. In Ad12 transformed rodent cells, the class I enhancer is down-regulated through increased binding of the repressor COUP-TF to the R2 element and decreased binding of the activator NF-kappa B (p50/p65) to the R1 element. The reduced surface levels of class I antigens contribute to the tumorigenic potential of Ad12 transformed cells by favoring their immunoescape from cytotoxic T-lymphocytes. Previous studies using transformed cells containing hybrid Ad5/Ad12 E1A (plus Ad12 E1B) genes have indicated that sequences within the first exon of the 266R Ad12 E1A gene are required for class I down-regulation and tumorigenesis. In this study we demonstrate that these same sequences, which exclude the Ad12 CR3 transactivation domain, are also required for increased COUP-TF binding to the R2 element and decreased NF-kappa B binding to the R1 element of the class I enhancer. We further show that diminished NF-kappa B binding is not due to a lack of NF-kappa B1-p50 in the nuclei of Ad12 transformed rat cells.

Negative regulation by the R2 element of the MHC class I enhancer in adenovirus-12 transformed cells correlates with high levels of COUP-TF binding.

The transcriptional down-regulation of the major histocompatibility complex (MHC) class I antigens in adenovirus type 12 (Ad12) transformed cells gives them the potential to escape immunosurveillance and to form tumors. The enhancer of the class I promoter is the target of transcriptional repression which is mediated by the E1A gene of Ad12. The R2 region within the class I enhancer acts as a negative element in Ad12-transformed cells and exhibits a stronger binding activity than is observed in nontumorigenic Ad5-transformed cells, which are not reduced in class I expression. The R2 element contains a nuclear hormone receptor half-site consensus sequence, AGGTCA, which is required for both the binding activity and the ability of R2 to act as a negative element in Ad12-transformed cells. In this study, we show that an orphan hormone receptor protein, COUP-TF, contributes to the differential R2 binding activity observed between Ad12- and Ad5-transformed cells. Additionally, COUP-TF was shown to bind as a dimer to the R2 element and to use the consensus AGGTCA as one half-site and its 3' flanking sequence as a probable second degenerate half-site. Since COUP-TF can act as a transcriptional repressor, we suggest that the higher COUP-TF binding activity to the R2 element in Ad12-transformed cells contributes to down-regulation of class I transcription and, consequently, tumorigenesis.

E1A-based determinants of oncogenicity in human adenovirus groups A and C.

A broad spectrum of genetic and molecular investigations carried out with group C, Ad2 and Ad5, and with group A, Ad12, have shown that early region1 (E1) gene products are sufficient for complete transformation of rodent cells in vitro by these viruses. During the past quarter century, the processes by which E1A proteins, in cooperation with E1B proteins, perturb the cell cycle and induce the transformed phenotype, have become well defined. Somewhat less understood is the basis for the differential oncogenicity of these two groups of viruses, and the processes by which the E1A proteins of Ad12 induce a tumorigenic phenotype in transformants resulting from infection of cells in vivo and in vitro. In this chapter we review previous findings and present new evidence which demonstrates that Ad12 E1A possesses two or more independent functions enabling it to induce tumors. One of these functions lies in its capacity to repress transcription of MHC class I genes, allowing the tumor cells to avoid lysis by cytotoxic T lymphocytes. We have shown that class I repression is mediated through increased binding of repressor COUP-TF and decreased binding of NF-kB to the class I enhancer. In addition to mediating immune escape, E1A also determines the susceptibility of transformants to Natural Killer (NK) cell lysis, and in this case, also, Ad12 transformants are not susceptible. By using Ad12 mutants containing chimeric E1A Ad12-Ad5 genes, point mutations, or a specific deletion, we have shown that the unique spacer region of Ad12 E1A is an oncogenic determinant, but is not required for transformation in vitro. Given that the E1A regions responsible for class I repression are first exon encoded, we have examined a set of cell lines transformed by these altered viruses, and have found that while they display greatly reduced tumorigenicity, they maintain a wildtype capacity to repress class I transcription. Whether the spacer contributes to NK evasion remains unresolved. Lastly, we discuss the properties of the Ad2/Ad5 E1A C-terminal negative modulator of tumorigenicity, and examine the effects on transformation, tumor induction and transformant tumorigenicity, when the Ad5 negative modulator is placed by chimeric construction in Ad12 E1A.

Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules.

Herein we outline three methods that, when coupled with cell-free protein synthesis, permit the identification of recombinant DNA molecules encoding specific polypeptides. RNAs enriched by fractionation on methylmercury hydroxide agarose gels are used to prepare sequence-specific probes. Recombinant DNA clones thus identified are further characterized as to their encoded polypeptides by either hybrid selection or hybrid arrest of translation.

The RITE assay: identifying effectors that target the transcription machinery using phage display technology.

We describe an approach using phage display to identify effectors (activators and repressors) of transcription based on the particular component of the general transcription machinery that they target. We refer to this approach as the reverse identification of transcriptional effectors (RITE) assay. A library of phages containing cDNA-encoded peptides displayed on their surfaces is screened using as the target a specific region of one of the general transcription factors (e.g., the C terminus of hTAFII135). The amino acid sequence encoded by the cDNA of an interacting phage is determined and analyzed in a database homology search to identify known or novel factors that may interact with the target protein. Candidate effectors from the homology search are synthesized from recombinant clones and tested for their abilities to bind to the target protein and to functionally modulate transcription in vivo when co-expressed with the transcriptional target protein. Because the RITE assay is a direct measure of the interactions between general transcription proteins and their effectors, it has an advantage over the well-known yeast two-hybrid system, which is not amenable to identifying transcription factor interactions.

Detailed kinetics of adenovirus type-5 steady-state transcripts during early infection.

The appearance and steady-state accumulation of specific viral RNAs during the early phase of adenovirus type 5 (Ad5) infection was examined. HeLa cells were synchronously infected and harvested at 30 min intervals throughout the first 12 h of infection. Total cytoplasmic RNA was extracted from infected cells and analyzed by hybridization-selection and translation to identify the viral mRNAs from each early region on the basis of the protein products they encode. The same RNA samples were used for S-1 nuclease and Northern blot analyses to quantitatively compare the levels of individual viral RNAs that accumulate within each early transcription region (E1A, E1B, L1, E2A, E3 and E4). The salient features of this analysis show that RNA accumulation occurs first from E1A followed by E2A, E3 and E4, E1B and lastly, L1. Although the profile of RNA accumulation was unique for each early region, overlapping RNAs within E1A, E3, and E4, respectively, remained generally parallel to one another throughout early infection, in contrast to RNAs from E1B and L1, respectively. Since both the appearance and quantitative accumulation of specific early viral mRNAs were examined at many time points, a number of subtleties associated with the complex dynamics of early Ad5 gene expression were revealed. In particular, the L1 region was shown to transcribe from the major late promoter two early RNAs of 3.81 Kb and 3.5 Kb, either or both of which encode the 52,55 kDa proteins; the auxiliary i leader sequence was found on the 3.81 Kb RNA but not on the 3.5 Kb RNA.