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Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford–AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

On 31 December 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases that was linked to a wet market in the city of Wuhan, China. The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus with similarity to bat coronaviruses. As of 28 January 2020, there are in excess of 4,500 laboratory-confirmed cases, with > 100 known deaths. As with the SARS-CoV, infections in children appear to be rare. Travel-related cases have been confirmed in multiple countries and regions outside mainland China including Germany, France, Thailand, Japan, South Korea, Vietnam, Canada, and the United States, as well as Hong Kong and Taiwan. Domestically in China, the virus has also been noted in several cities and provinces with cases in all but one provinence. While zoonotic transmission appears to be the original source of infections, the most alarming development is that human-to-human transmission is now prevelant. Of particular concern is that many healthcare workers have been infected in the current epidemic. There are several critical clinical questions that need to be resolved, including how efficient is human-to-human transmission? What is the animal reservoir? Is there an intermediate animal reservoir? Do the vaccines generated to the SARS-CoV or MERS-CoV or their proteins offer protection against 2019-nCoV? We offer a research perspective on the next steps for the generation of vaccines. We also present data on the use of in silico docking in gaining insight into 2019-nCoV Spike-receptor binding to aid in therapeutic development. Diagnostic PCR protocols can be found at https://www.who.int/health-topics/coronavirus/laboratory-diagnostics-for-novel-coronavirus.

Lesbian, gay, bisexual, trans, other queer, and Two-Spirit (LGBTQ2+) people are particularly at risk for the psycho-social consequences of the COVID-19 pandemic, though population-tailored research within this context remains limited. This study examines the extent of, and associations between, increased alcohol and cannabis use and deteriorating mental health among LGBTQ2+ adults in Canada during the COVID-19 pandemic. Data are drawn from LGBTQ2+ respondents to a repeated, cross-sectional survey administered to adults living in Canada (May 2020–January 2021). Bivariate cross-tabulations and multivariable logistic regression models were utilized to examine associations between increased alcohol and cannabis use, and self-reported mental health, overall coping, and suicidal thoughts. Five-hundred and two LGBTQ2+ participants were included in this analysis. Of these, 24.5% reported increased alcohol use and 18.5% reported increased cannabis use due to the pandemic. In the adjusted analyses, increased alcohol use was associated with poor overall coping (OR = 2.28, 95% CI = 1.28–4.07) and worse self-reported mental health (OR = 1.98, 95% CI = 1.21–3.25), whereas increased cannabis use was associated with suicidal thoughts (OR = 2.30, 95% CI = 1.16–4.55). These findings underscore the need for population-tailored, integrated substance use and mental health supports to address interrelated increases in alcohol/cannabis use and worsening mental health among LGBTQ2+ adults, in the context of the COVID-19 pandemic and beyond.

SummaryCas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9’s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Summary Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9’s activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Oncolytic virotherapy represents an emerging development in anticancer therapy. Although it has been tested against a variety of cancers, including breast cancer, the efficacy of oncolytic viral vectors delivered as a monotherapy is limited. Enhancing viral oncolytic therapies through combination treatment with anticancer agents is a feasible strategy. In this study, we considered a chemovirotherapeutic approach for treating breast adenocarcinoma using oncolytic measles virus (MV) and the chemotherapeutic agent camptothecin (CPT). Our results demonstrated that co-treatment of MV with CPT yielded enhanced cytotoxicity against breast cancer cells. Low dosage CPT combined with MV was also found to elicit the same therapeutic effect as high doses of CPT. At the lower dosage used, CPT did not inhibit the early stages of MV entry, nor reduce viral replication. Further studies revealed that co-treatment induced significantly enhanced apoptosis of the breast cancer cells compared to either MV or CPT alone. Overall, our findings demonstrate the potential value of MV plus CPT as a novel chemovirotherapeutic treatment against breast cancer and as a strategy to enhance MV oncolytic activity.

The inhibitors carbobenzoxy (Z)- d -Phe- l -Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z- d -Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z- d -Phe- l -Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z- d -Phe- l -Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles “blind” to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces.

Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.

Without a vaccine, hepatitis C virus (HCV) remains a global medical and socio-economic burden, predisposing about 170 million carriers worldwide to end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Although the recently developed direct-acting antivirals (DAAs) have revolutionized hepatitis C treatment, most of them are unsuitable for monotherapy due to risks of resistance, thus necessitating combination with interferon (IFN)-alpha, ribavirin, or additional DAAs. More importantly, the high cost associated with the DAAs restricts their accessibility to most parts of the world. Developing novel cost-effective anti-HCV therapeutics may help expand the scope of antivirals and treatment strategies against hepatitis C. Herein, we applied an activity-based and fraction-guided analysis of extracts from the medicinal plant Phyllanthus urinaria (P. urinaria), which yielded fraction 13 (F13) as possessing the most potent inhibitory activity against early viral entry of cell-culture HCV infection. Chemical analysis (silica gel chromatography followed by ESI LC-MS plus (1)H and (13)C NMR) of F13 identified loliolide (LOD), a monoterpenoid lactone, as a novel inhibitor of HCV entry. Specifically, LOD could efficiently inactivate HCV free virus particles, abrogate viral attachment, and impede viral entry/fusion, with minimal effect on viral replication/translation, particle production, and induction of type I IFN host antiviral immune response. ELISA-based binding analysis confirmed the monoterpenoid's ability in efficiently blocking HCV particle attachment to the host cell surface. Furthermore, LOD could inhibit infection by several genotypic strains of HCV. This is the first report characterizing P. urinaria and its bioactive compound LOD as potent HCV entry inhibitors, which merit further evaluation for development as candidate antiviral agents against hepatitis C.

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.

Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.

PVRL4 (nectin-4) was recently identified as the epithelial receptor for members of the Morbillivirus genus, including measles virus, canine distemper virus and peste des petits ruminants virus. Here, we describe the role of PVRL4 in morbillivirus pathogenesis and its promising use in cancer therapies. This discovery establishes a new paradigm for the spread of virus from lymphocytes to airway epithelial cells and its subsequent release into the environment. Measles virus vaccine strains have emerged as a promising oncolytic platform for cancer therapy in the last ten years. Given that PVRL4 is a well-known tumor-associated marker for several adenocarcinoma (lung, breast and ovary), the measles virus could potentially be used to specifically target, infect and destroy cancers expressing PVRL4.

Background & Aims A vaccine against hepatitis C virus (HCV) is unavailable and cost-effective antivirals that prevent HCV infection and re-infection, such as in the transplant setting, do not exist. In a search for novel and economical prophylactic agents, we examined the antiviral activity of saikosaponins (SSa, SSb2, SSc, and SSd) from Bupleurum kaoi root (BK) as entry inhibitors against HCV infection. Methods Infectious HCV culture systems were used to examine the effect of saikosaponins on the complete virus life cycle (entry, RNA replication/translation, and particle production). Antiviral activity against various HCV genotypes, clinical isolates, and infection of primary human hepatocytes were also evaluated. Results BK and the saikosaponins potently inhibited HCV infection at non-cytotoxic concentrations. These natural agents targeted early steps of the viral life cycle, while leaving replication/translation, egress, and spread relatively unaffected. In particular, we identified SSb2 as an efficient inhibitor of early HCV entry, including neutralization of virus particles, preventing viral attachment, and inhibiting viral entry/fusion. Binding analysis, using soluble viral glycoproteins, demonstrated that SSb2 acted on HCV E2. Moreover, SSb2 inhibited infection by several genotypic strains and prevented binding of serum-derived HCV onto hepatoma cells. Finally, treatment with the compound blocked HCV infection of primary human hepatocytes. Conclusions Due to its potency, SSb2 may be of value for development as an antagonist of HCV entry and could be explored as prophylactic treatment during the course of liver transplantation.

Human torovirus (HTV) and Breda virus (BRV), members of the genus torovirus in the family Coronaviridae, are established infectious agents of humans and cattle, respectively. The hemagglutinin-esterase (HE) gene of Breda virus serotype 2 (BRV-2) has been identified and the nucleotide sequence for BRV serotype 1 (BRV-1) genome which contains the open reading frames for the viral structural proteins has been reported revealing the presence of a 1.25 kb gene whose nucleotide sequence is identical to that of the BRV-2 HE gene. In this study, we amplified the 1.2kb HE gene from the HTV genome using long RT-PCR and sequenced the amplicon directly. At the nucleotide level, the HTV HE gene manifests 85% sequence identity to the HE genes of BRV-1 and BRV-2 and 89% identity with the X pseudogene sequence of BEV. The 1.25 kb amplicons which contained the HE genes of BRV-1 and HTV were cloned and expressed in a baculovirus system and the proteins purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Hyperimmune sera prepared in guinea pigs against these proteins were reactive with both bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoblot, they reacted specifically with a 65 kDa protein corresponding in size to the torovirus HE protein. Furthermore, the hyperimmune sera but not the preimmune sera reacted with a series of BTV-positive and HTV-positive fecal specimens by immunoblot and dot blot analysis. By immunoelectron microscopy (IEM) torovirus particles from BTV-positive specimens from calves with diarrhea and HTV-positive specimens from patients were aggregated by the hyperimmune sera. Human convalescent sera and gnotobiotic calf post-infection sera reacted by immunoblot with the expressed 65 kDa protein. The expressed HE protein of HTV has important diagnostic potential.

Group B coxsackieviruses are etiologically linked with many human diseases including acute myocarditis and associated chronic dilated cardiomyopathy. Well-established CVB3 cardiovirulent strains (CVB3c (s) ) with known phenotypic differences have been used to study the pathogenesis of virus-induced heart disease. The receptor-binding characteristics of cardiovirulent CVB3 are not known, but may represent one mechanism accounting for differences in disease virulence. In this study, interactions between CVB3c (s) and the decay-accelerating factor (DAF or CD55) cell surface receptor were examined. Anti-DAF monoclonal antibodies (MAbs) blocked virus binding and infection of susceptible HeLa cells. Virus binding was significantly reduced by treatment of these cells with phosphatidylinositol phospholipase C enzyme, which rendered them DAF-deficient. CVB3c (s) exhibited a differential propensity for the DAF receptor, as several cardiovirulent strains interacted more strongly than others. However, virus binding and infection was always most effectively blocked by MAbs directed against the SCR 2 and 3 domains of DAF, suggesting that binding occurs at a similar site(s) on the molecule for all strains. Virus binding and internalization were associated with DAF down-regulation at the cell surface, as monitored by flow cytometry analysis. Cardiovirulent CVB3 did not interact with molecules functionally and/or structurally related to DAF, including CD35, CD46, Factor H, or C4-binding protein. Adenovirus type 2 (Ad2) does not use the DAF receptor. However, competitive binding assays between Ad2 and CVB1-6, CVB3c (s) , anti-DAF MAbs, or DAF-reduced cells indicated that DAF is associated with Ad2 receptors on the HeLa cell membrane. In summary, this study indicates that DAF is an attachment receptor for cardiovirulent CVB3 and that DAF interaction may be important in the pathogenesis of CVB-mediated heart disease.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its α-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis.

Anti-CD18 monoclonal antibodies (MAb) have demonstrated variable protection against neutrophil (PMN)-mediated myocardial reperfusion injury. To identify factors contributing to this variability, open-chest dogs underwent coronary artery occlusion for 90 min followed by reperfusion for 3.5 h. Ten minutes before reperfusion the dogs received saline (n = 18) or one of three anti-CD18 MAb: MHM.23, R15.7, or PLM-2 (2, 1, and 1 mg/kg and n = 19, 8, and 4, respectively). Collateral flow was measured with radioactive microspheres, area at risk was assessed with monastral blue dye, and infarct size was measured postmortem by triphenyltetrazolium chloride. In vitro, all three MAb bound to canine PMNs, but only MHM.23 and R15.7 inhibited their adherence to keyhole limpet hemocyanin-coated plastic. In vivo, only MHM.23 and R15.7 significantly reduced infarct size after adjusting for the effect of collateral flow. MHM.23 afforded protection in dogs with moderate ischemia (epicardial collateral flow > 0.1 ml.min-1.g-1, infarct size reduced 46%) but not in dogs with more severe ischemia. Only R15.7 was effective in dogs with severe ischemia. Although MHM.23 and R15.7 produced similar inhibition of tissue PMN accumulation, as reflected by myeloperoxidase activity. R15.7 markedly inhibited H2O2 production by PMNs after exposure to platelet-activating factor, whereas MHM.23 had only a minimal effect. The effectiveness of different anti-CD18 MAb in preventing reperfusion injury appears to be 1) highly dependent on the specific anti-CD18 MAb employed, 2) predicted only partially by in vitro binding to PMNs, static in vitro tests of PMN adherence, or the extent of inhibition of PMN accumulation in vivo, 3) related more to their ability to inhibit oxidant release from activated PMNs, and 4) strongly influenced by the severity of myocardial ischemia before reperfusion.

This review takes a general approach to describing host cell factors that facilitate measles virus (MeV) infection and replication. It relates our current understanding of MeV entry receptors, with emphasis on how these host cell surface proteins contribute to pathogenesis within its host. The roles of SLAM/CD150 lymphocyte receptor and the newly discovered epithelial receptor PVRL4/nectin-4 are highlighted. Host cell factors such as HSP72, Prdx1, tubulin, casein kinase, and actin, which are known to impact viral RNA synthesis and virion assembly, are also discussed. Finally the review describes strategies used by measles virus to circumvent innate immunity and confound the effects of interferon within the host cell. Proteomic studies and genome wide RNAi screens will undoubtedly advance our knowledge in the future.

Measles virus normally infects only primate cells. The receptor for measles virus has recently been shown to be the complement regulator CD46, also known as membrane cofactor protein. Transfection of rodent cells with human CD46 renders them susceptible to the virus, suggesting that transgenic animals may prove useful for testing antiviral agents and vaccines.

The solid phase synthesis of 3′ deoxyoligonucleotide (17 mer)-peptide conjugates is described. The oligonucleotide is complementary to the template strand of the measles virus in the region of the nucleocapsid protein on the 60 nucleotide leader RNA. The conjugates were synthesized on teflon support using a combination of solid phase FMOC peptide chemistry and phosphoramidite chemistry linked by a branched modifier.

The entomopoxviruses (insect poxviruses) of eastern spruce budworm (Choristoneura fumiferana), two year cycle spruce budworm (C. biennis) and the Indian red army worm (Amsacta moorei) are being studied in our laboratory for their potential as biological insecticides and expression vectors. These viruses characteristically replicate in the cytoplasm of insect cells and produce occlusion bodies that serve to protect the virion from the environment. By analogy to mammalian pox-viruses, they should also contain a viral thymidine kinase (TK) that functions in viral DNA synthesis. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analogue and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses were identified, cloned and sequenced. The sequences of the TK genes of the entomopoxviruses were closely related and exhibited 63.2% identity and 9.9% similarity at the protein level. However, there was only 36.7% identity and 13.6% similarity when these enzymes were compared to their mammalian poxvirus counterpart in vaccinia virus. Finally, one entomopoxvirus TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. The results presented in this paper provide impetus for the design of a recombinant entomopoxvirus expression system in which foreign genes could be introduced into the viral TK locus under selective pressure from bromodeoxyuridine.

In order to be infectious, viruses must possess a mechanism allowing their nucleic acid to pass through the plasma membrane of the target cells. Two such mechanisms have been described for en- veloped viruses, both requiring membrane fusion. One path is by direct fusion of the viral membrane with the plasma membrane of the target cell, while the other path requires prior endocytosis of the virus through coated pits, followed by fusion of the viral membrane with an endosomal membrane of the host cell. The latter mechanism requires a lowering of the pH, while the former can take place at physio- logical pH. In the present study, we have chosen measles virus as an example of a virus that fuses to the plasma membrane and influenza virus as one requiring endosomal acidification. There has been much work attempting to eluci- date the mechanism of viral fusion to target mem- branes. Through experiments of proteolytic cleav- age, mutagenesis, and reconstitution of viral proteins into virosomes, it is well established that the F pro- tein of measles and the

Entomopoxviruses replicate in the cytoplasm of insect cells and characteristically produce occlusion bodies which serve to protect the virion from the environment; the major component of these bodies is a protein called spheroidin. We have previously identified and sequenced the gene encoding the major occlusion body protein of eastern spruce budworm (Choristoneura biennis) entomopoxvirus (CbEPV) and found it to encode a 47K polypeptide which aggregates due to the formation of intermolecular disulphide bonds. In this publication we demonstrate that the insect poxvirus of Amsacta moorei produces spheroidin with a unit Mr of 114.8K. The gene for this protein was cloned and sequenced, and the predicted polypeptide was demonstrated to contain 38 cysteine residues, a leucine zipper for possible protein-protein interactions and 14 potential Asn-linked glycosylation sites. Other than possessing a large number of sulphydryl groups, this protein showed no homology to its analogue found in cells infected with CbEPV. Antibodies directed against occlusion body proteins of the two viruses also failed to cross-react significantly on Western blots. In addition, nucleic acid probes prepared from the two different genes did not cross-hybridize on Southern blots of genomic DNA prepared from the viruses. Finally, the occlusion body proteins from the two insect viruses were compared with the A-type inclusion body protein of cowpox virus. Again, little homology between these proteins was evident, with the exception of a generally high cysteine content and a similarity between their late gene promoters. We conclude that the major occlusion body proteins of different poxviruses possess diverse primary structures, but all are capable of yielding large aggregates through the formation of disulphide bonds.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.

31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.

The solid phase synthesis of 3′ deoxyoligonucleotide (17 mer)-peptide conjugates is described. The oligonucleotide is complementary to the template strand of the measles virus in the region of the nucleocapsid protein on the 60 nucleotide leader RNA. The conjugates were synthesized on teflon support using a combination of solid phase FMOC peptide chemistry and phosphoramidite chemistry linked by a branched modifier.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

A transgenic mouse containing the complete human SLAM (hSLAM/CD150) gene, including its endogenous promoter for transcription, was generated by using human genomic DNA cloned into a bacterial artificial chromosome. hSLAM, the primary receptor for measles viruses (MV), was expressed on activated B, T, and dendritic cells with an expression profile equivalent to that of humans. We demonstrated that hSLAM + cells obtained from the transgenic mouse, including activated B, T, and dendritic cells, were susceptible to MV infection in a receptor-dependent manner. Evidence was provided for transient infection in the nasal lymph nodes of hSLAM + mice after intranasal inoculation. Virus was rapidly cleared without signs of secondary replication. To improve the efficiency of MV production, the hSLAM + mice were bred with mice having a Stat1 -deficient background. These mice were more susceptible to MV infection and produced more virus particles. After intranasal and intraperitoneal inoculation of these mice with MV, infections of the thymus, spleen, nasal, mesenteric, and leg lymph nodes were detected. Upon necropsy, enlarged lymph nodes and spleen were apparent. Flow cytometric analysis showed that abnormally large numbers of mature neutrophils and natural killer cells caused the splenomegaly. The hSLAM transgenic mouse constitutes an improved rodent model for studying the interaction of MV with immune cells that more accurately reflects the infection pattern found in humans.

Natural isolates of measles virus readily infect several lymphocyte cell lines. These viruses appear to use a receptor other than CD46, the molecule to which most laboratory strains of virus bind. Methods used to identify and characterize this lymphocyte receptor for measles virus are described in this study. A binding assay with a soluble form of measles virus H protein demonstrated that B-cell lines, activated with Epstein–Barr virus, or T cells, transformed with human T-cell leukemia virus, exhibit this receptor on their cell surfaces. On the other hand, resting lymphocytes, monocytes, or immature leukocytes either failed to express or possessed reduced levels of this receptor. A cDNA library derived from B95-8 marmoset B-cell lines was used to identify this receptor through expression cloning. This molecule was shown to be CDw150, which is also known as the signaling lymphocytic activation molecule (SLAM). When the lymphocyte receptor was expressed in Chinese hamster ovary (CHOP) or human embryonic kidney (293T) cells, these cells became susceptible to lymphotropic as well as laboratory strains of measles virus. Binding assays confirmed that either lymphotropic or laboratory strains of measles virus could adhere to human or marmoset CDw150, but interaction with the mouse homolog was weak. These infections were independent of the presence of CD46 on the host cell surface. Interaction of measles virus with CDw150(SLAM) could explain the immunosuppressive properties of this virus.

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.

Summary Measles virus normally causes disease in human beings, and the host range of this virus may be determined by a specific receptor on the surface of primate cells. Human-rodent somatic cell hybrids were tested for their ability to bind measles virus, and only cells that contained human chromosome 1 were capable of binding virus. A study of lymphocyte markers suggested that the complement regulator known either as membrane cofactor protein or CD46 was the measles virus receptor. We proved this hypothesis by demonstrating that hamster cell lines that expressed human CD46 could subsequently bind virus. Furthermore, infected CD46 + cells produced syncytia and viral proteins. Finally, polyclonal antisera against CD46 inhibited virus binding and infection. These results prove that human CD46 permits cells both to bind measles virus and to support infection.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.

The development of canarypox virus (CPV) recombinants expressing the hemagglutinin (HA) and fusion (F) glycoproteins of measles virus (MV) is described. Inoculation of the CPV-MV recombinants into avian or nonavian tissue culture substrates led to the expression of authentic MVF and MVHA as determined by radioimmunoprecipitation and surface immunofluorescence. In contrast to avian-derived tissue culture, no productive replication of the CPV recombinant was evident in tissue culture cells derived from nonavian origin. On inoculation of dogs, a species restricted for avipoxvirus replication, the recombinants elicited a protective immune response against a lethal canine distemper virus (CDV) challenge. The level of MV neutralizing antibodies and the level of protection induced against CDV challenge achieved by the host-restricted CPV vector were equivalent to that obtained by vaccinia virus vectors expressing the same MV antigens.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.

About 100 nucleotides of DNA sequence at the 5' noncoding region of the Choristoneura biennis entomopoxvirus spheroidin gene was chemically synthesized and inserted into a vaccinia expression vector, interrupting the vaccinia thymidine kinase gene. When the bacterial beta-galactosidase gene was introduced downstream of this sequence and a recombinant vaccinia virus containing these inserts was obtained by homologous recombination, beta-galactosidase was shown to be expressed at a high level late in the vaccinia infection cycle. The level of beta-galactosidase expression was four- to fivefold higher with this spheroidin-vaccinia recombinant virus than with a similar recombinant in which the beta-galactosidase gene was under the control of the vaccinia 7.5-kDa promoter. Primer extension and S1 mapping of the 5' terminus of the beta-galactosidase transcript located the transcription initiation site within the spheroidin DNA sequence, confirming the promoter nature of this DNA sequence in the vaccinia system. Dot blot analysis indicated that the difference in beta-galactosidase expression with these two recombinant viruses can be attributed to the difference in their transcript levels. We also demonstrated that full promoter activity encoded in the spheroidin 5' noncoding sequence was contained within a 38-nucleotide DNA fragment.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.

The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and its functions from the VSV polymerase.

Previous studies from this laboratory showed that oligopeptides with amino acid sequences similar to the sequence of the N-terminal region of the F1 polypeptide of paramyxoviruses inhibited the membrane fusing activity of the F protein, and thereby inhibited virus infectivity at the level of penetration and virus-induced cell fusion and hemolysis. The site of action of these oligopeptide inhibitors has been investigated. Radioactively labeled oligopeptides were found to bind to cells, but not to virus. Pretreatment of cells, but not virus, at 4 degrees with oligopeptides inhibited the initiation of infection and hemolysis induced by measles virus. The binding of the oligopeptides to cells was reversible at 25 or 37 degrees. Oligopeptides were synthesized with a chloromethylketone group to enable them to bind irreversibly, or with an azido group to permit them to be cross-linked in situ by photoactivation. The results with these derivatized oligopeptides, which retained their inhibitory activity, confirmed that they bind to, and express their inhibitory activity on, cells and not virus. The results suggest that the oligopeptides react with receptor sites on the cell membrane and inhibit membrane-fusing activity by competing with the F1 polypeptide for such sites. A Scatchard analysis of the binding of an oligopeptide to CV-1 cells revealed that it bound with a dissociation constant of 1.2 X 10(-7) M and that there were approximately 3.0 X 10(6) binding sites per cell.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.

Group B coxsackieviruses are etiologically linked to many human diseases, and cell surface receptors are postulated to play an important role in mediating their pathogenesis. The coxsackievirus adenovirus receptor (CAR) has been shown to function as a receptor for selected strains of coxsackievirus group B (CVB) serotypes 3, 4, and 5 and is postulated to serve as a receptor for all six serotypes. In this study, we demonstrate that CAR can serve as a receptor for laboratory reference strains and clinical isolates of all six CVB serotypes. Infection of CHO cells expressing human CAR results in a 1000-fold increase in CVB progeny virus titer compared to mock transfected cells. CAR was shown to be a functional receptor for swine vesicular disease virus (SVDV), as CHO-CAR cells but not CHO mock transfected controls were susceptible to SVDV infection, produced progeny SVDV, and developed cytopathic effects. Moreover, SVDV infection could be specifically blocked by monoclonal antibody to CAR (RmcB). SVDV infection of HeLa cells was also inhibited by an anti-CD55 MAb, suggesting that this virus, like some CVB, may interact with CD55 (decay accelerating factor) in addition to CAR. Finally, pretreatment of CVB or SVDV with soluble CAR effectively blocks virus infection of HeLa cell monolayers.

A cDNA library was constructed from poly(A)+ RNA prepared from VERO cells infected with the Edmonston strain of measles virus. Clones corresponding to five major viral-specific transcripts were identified by northern blot hybridization analysis. Probes prepared from these five clones detected an additional five minor viral RNA transcripts. The sizes and hydridization patterns of these minor RNA species are consistent with their being bicistronic transcripts arising from a viral genomic template with the gene order 3’. NP-P/C-M-F-HA-(L).. 5’. To assess the coding capacity of these cDNA clones they were inserted into pSP64, transcribed in vitro, the RNA was translated in reticulocyte lysates, and the protein was immunoprecipitated with specific antisera. From this analysis the genes for NP, P/C, M, F, and HA were identified. In vitro translation of natural mRNAs and SP6 transcripts of cDNAs consistently produced smaller polypeptides that appear to be initiated at internal AUGs. The relative abundance of these various cell-free translation products reflects the probability of translational initiation at the various in-frame AUGs. The patterns observed suggest that other factors besides sequences immediately flanking the AUGs have a significant effect on the selection of translational initiation sites. An increase in translational efficiency of the F transcript was achieved by removing 450 bases of the G-C-rich 5’ noncoding region.

The entirety of the phosphoprotein gene of measles virus has been sequenced. The gene is composed of 1,657 nucleotides and specifies a 507-amino-acid protein (P). A second overlapping reading frame was predicted from the sequence and specifies a 186-amino-acid protein (C). Through the use of antisynthetic peptide antibodies, we show that both proteins are expressed in virally infected cells. Both proteins are expressed from a functionally bicistronic mRNA through independent initiation of ribosomes at the respective AUG codons. Using immunofluorescent microscopy, we localized the C protein in the nucleus and in cytoplasmic inclusions within the infected cells.

The F glycoprotein of paramyxoviruses is responsible for cell fusion and hemolysis and for virus penetration via fusion of viral and cell membranes. These functions are activated by specific proteolytic cleavage of an inactive precursor (F0) into two disulfide-linked polypeptides (F1 and F2). The susceptibility of the F0 protein to cleavage by a host protease is a major determinant of virus host range and virulence. Synthetic oligopeptides that mimic the N-terminal region of the F1 polypeptide are specific inhibitors of paramyxoviruses, and oligopeptides that mimic the N-terminus of the HA2 polypeptide of influenza virus, also generated by cleavage, specifically inhibit that virus. Antibodies to F protein prevent the spread of paramyxovirus infection via membrane fusion, but antibodies to HN protein do not, although they neutralize released virus. These results and previous findings that formalin-treated virus does not induce antibodies to F protein provide an explanation for atypical measles. The HN protein has both receptor-binding and neuraminidase activities, and Cl- inhibition of neuraminidase may modulate these antagonistic activities. Studies in patients with subacute sclerosing panencephalitis (SSPE) suggest that there is a host restriction of synthesis of the M protein of measles virus in brain cells which is involved in the abortive, persistent infection that causes SSPE.