CUL4A silencing attenuates cervical carcinogenesis and improves Cisplatin sensitivity.

CUL4A is an ubiquitin ligase deregulated in numerous pathologies including cancer and even hijacked by viruses for facilitating their survival and propagation. However, its role in Human papilloma virus (HPV)-mediated cervical carcinogenesis remains elusive. The UALCAN and GEPIA datasets were analyzed to ascertain the transcript levels of CUL4A in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients. Subsequently, various biochemical assays were employed to explore the functional contribution of CUL4A in cervical carcinogenesis and to shed some light on its involvement in Cisplatin resistance in cervical cancer. Our UALCAN and GEPIA datasets analyses reveal elevated CUL4A transcript levels in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients that correlate with adverse clinicopathological parameters such as tumor stage and lymph node metastasis. Kaplan-Meier plot and GEPIA assessment depict poor prognosis of CESC patients having high CUL4A expression. Varied biochemical assays illustrate that CUL4A inhibition severely curtails hallmark malignant properties such as cellular proliferation, migration, and invasion of cervical cancer cells. We also show that CUL4A knockdown in HeLa cells causes increased susceptibility and better apoptotic induction toward Cisplatin, a mainstay drug used in cervical cancer treatment. More interestingly, we find reversion of Cisplatin-resistant phenotype of HeLa cells and an augmented cytotoxicity towards the platinum compound upon CUL4A downregulation. Taken together, our study underscores CUL4A as a cervical cancer oncogene and illustrates its potential as a prognosis indicator. Our investigation provides a novel avenue in improving current anti-cervical cancer therapy and overcoming the bottle-neck of Cisplatin resistance.

Direct Correlation between the Secondary Structure of an Amphiphilic Polymer and Its Prominent Antiviral Activity.

An amphiphilic segmented polyurethane (F-PU-S), with pendant sulfate groups and a flexible hydrocarbon backbone, exhibits intrachain H-bonding-reinforced folding and hierarchical assembly, producing an anionic polymersome with efficient display of sulfate groups at the surface. It shows an excellent antiviral activity against Sendai virus (SV) by inhibiting its entry to the cells. Mechanistic investigation suggests fusion of the SV and the polymersome to produce larger particles in which neither the folded structure of the polymer nor the fusogenic property of the SV exists anymore. In sharp contrast, a structurally similar polymer R-PU-S, in which the chain folding pathway is blocked by replacing the flexible C6 chain with a rigid cyclohexane chain in the backbone, cannot form a similar polymersome structure and hence does not exhibit any antiviral activity. On the other hand, the third polymer (F-PU-C), which is similar to F-PU-S except for the pendant anionic groups (carboxylate instead of sulfate), also fails to exhibit any antiviral activity against SV, confirming the essential role of the chain folding as well as the pendant sulfate groups for the fusion-induced antiviral activity of F-PU-S, which provides an important structural guideline for developing new antiviral polymers.

Analysis of the dark proteome of Chandipura virus reveals maximum propensity for intrinsic disorder in phosphoprotein.

Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.

Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze-Thaw Method.

Understanding the structure-function of inclusion bodies (IBs) in the last two decades has led to the development of several mild solubilization buffers for the improved recovery of bioactive proteins. The recently developed freeze-thaw-based inclusion body protein solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness. The present report investigates the reproducibility, efficiency, and plausible mechanism of the freeze-thaw-based IB solubilization. The percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in Escherichia coli and the quality attributes associated with the freeze-thaw-based solubilization method were analyzed in detail. The overall yield of the purified hGH and L-asparaginase protein was found to be around 14 and 25%, respectively. Both purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analyses revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides insight into the advantages, disadvantages, and molecular-structural information associated with the freeze-thaw-based solubilization method.

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV.

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.

Site-specific phosphorylation of villin remodels the actin cytoskeleton to regulate Sendai viral glycoprotein-mediated membrane fusion.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes.

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion-mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206-an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

Induction of Transcriptional Gene Silencing by Expression of shRNA Directed to c-Myc P2 Promoter in Hepatocellular Carcinoma by Tissue-Specific Virosomal Delivery.

Double-stranded RNA-mediated transcriptional gene silencing (TGS) has shown promising results over posttranscriptional gene silencing (PTGS) due to its long term and heritable nature. Various research groups have shed light on different mechanisms by which TGS operate. Some of these include histone modification, DNA methylation, or restriction of RNA polymerase binding onto the target gene's promoter. This serves as an added advantage since permanent c-Myc inactivation is critical for suppressing hepatocellular carcinoma (HCC). Inability to target cancer cells specifically, without affecting the normal cells, has been one of the biggest drawbacks of an effective cancer therapy. Therefore, we aimed to overcome this barrier by first generating tumor-specific transcriptional units expressing TGS inducing shRNAs against c-Myc's P2 promoter only in neoplastic liver cells. Secondly, we coupled this TGS inducing system with Sendai fusion virosomes for liver-specific delivery to minimize nonspecific side effects in vitro.

A novel placental like alkaline phosphatase promoter driven transcriptional silencing combined with single chain variable fragment antibody based virosomal delivery for neoplastic cell targeting [corrected].

BACKGROUND: Placental like alkaline phosphatase (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues [corrected]. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting. METHODS: PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties. RESULTS: Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region. CONCLUSION: A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.

Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.

Combination of hepatocyte specific delivery and transformation dependent expression of shRNA inducing transcriptional gene silencing of c-Myc promoter in hepatocellular carcinoma cells.

BACKGROUND: A specific targeting modality for hepatocellular carcinoma (HCC) could ideally encompass a liver cell specific delivery system of a transcriptional unit that is active only in neoplastic cells. Sendai virosomes, derived from Sendai viral envelopes, home to hepatocytes based on the liver specific expression of asialoglycoprotein receptors (ASGPRs) which are recognized by the Sendai virosomal fusion (F) proteins. As reported earlier by us and other groups, transcriptional gene silencing (TGS) does not require continuous presence of the effector siRNA/shRNA molecule and is heritable, involving epigenetic modifications, leading to long term transcriptional repression. This could be advantageous over conventional gene therapy approaches, since continuous c-Myc inactivation is required to suppress hepatocarcinoma cells. METHODS: Exploiting such virosomal delivery, the alpha-fetoprotein (AFP) promoter, in combination with various tumour specific enhancers, was used to drive the expression of shRNA directed against ME1a1 binding site of the proto-oncogene c-Myc P2 promoter, in order to induce TGS in neoplastic liver cells. RESULTS: The dual specificity achieved by the Sendai virosomal delivery system and the promoter/enhancer guided expression ensured that the shRNA inducing TGS was active only in liver cells that had undergone malignant transformation. Our results indicate that such a bimodal therapeutic system induced specific activation of apoptosis in hepatocarcinoma cells due to heterochromatization and increased DNA methylation of the CpG islands around the target loci. CONCLUSIONS: The Sendai virosomal delivery system, combined with AFP promoter/enhancer expression machinery, could serve as a generalized mechanism for the expression of genes deleterious to transformed hepatocarcinoma cells. In this system, the epigenetic suppression of c-Myc could have an added advantage for inducing cell death in the targeted cells.

Inhibition of the interaction between NS3 protease and HCV IRES with a small peptide: a novel therapeutic strategy.

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule.

Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Reciprocal regulation of AKT and MAP kinase dictates virus-host cell fusion.

Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

UNLABELLED: Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. CONCLUSION: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes.

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Association between human leukocyte antigen class II alleles and human papillomavirus-mediated cervical cancer in Indian women.

We investigated the association of human leukocyte antigen (HLA) II (DRB1 and DQB1) alleles with susceptibility to human papillomavirus (HPV)-associated cervical precancer and cancer cases in a hospital-based case-control study in a northern Indian population. A total of 202 subjects, including 100 patients comprising 31 cervical precancer (cervical intraepithelial neoplasia [CIN] 2/3) and 69 invasive cervical cancer cases, and 102 healthy controls participated in the study. Both patients and controls were screened for HPV infection using a polymerase chain reaction (PCR-based approach. Low-resolution PCR-sequence specific priming (PCR-SSP) was used to genotype HLA II (DRB1 and DQB1). Our results demonstrate that the DRB1*15 allele/DRB1*15-DQB1*06 haplotype may have a predisposition for HPV infection (p(c) < 0.05) or cervical cancer/precancer (p(c) < 0.05) development, whereas the DRB1*04 allele/DRB1*04-DQB1*03 haplotype might exhibit susceptibility to cervical precancerous lesions (p(c) < 0.05). The DRB1*13 allele/DRB1*13-DQB1*06 haplotype was strongly protective against risk to HPV infection (p(c) < 0.002) as well as cervical cancer (p(c) 0.01). Therefore, we have demonstrated that HLA DR-DQ polymorphisms are involved in genetic susceptibility to cervical cancer or HPV infection in a northern Indian population.

A histidine switch in hemagglutinin-neuraminidase triggers paramyxovirus-cell membrane fusion.

Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine "switch" in HN that triggers fusion.