William R. Gallaher

Membrane Interface-Interacting Sequences within the Ectodomain of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Putative Role during Viral Fusion

ABSTRACT We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH2-DKWASLWNWFNITNWLWYIK-CONH2(HIVc), the membrane interface-partitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2(HIVn), the fusion peptide at the N terminus of the subunit. Both HIVc and HIVn were seen to induce membrane fusion and permeabilization, although lower doses of HIVc were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIVc and HIVn were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIVc ability to induce membrane fusion or form complexes with HIVn but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surface-residing residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.

Th1/Th2 cytokine expression in saliva of HIV-positive and HIV-negative individuals : A pilot study in HIV-positive individuals with oropharyngeal candidiasis

Current data suggest that T-helper (Th)2-type cytokine responses are often associated with progression to AIDS in HIV-positive individuals. Similarly, Th2-type cytokines are associated with susceptibility to mucosal candidiasis, of which oropharyngeal candidiasis (OPC) is one of the most common opportunistic infections in HIV-positive individuals. Although little information is available on host defense mechanisms at the level of the oral mucosa, recent studies suggest that local cell-mediated immunity (CMI) is equally or more important than that in the periphery for host defense against mucosal Candida albicans infections. This study investigated the potential presence of oral-associated CMI through the expression of Th1/Th2-type cytokines in saliva of immunocompetent and immunocompromised individuals with and without OPC. Results showed a constitutive mixed Th1/Th2 cytokine expression (Th0) in whole saliva of healthy HIV-negative individuals. In contrast, HIV-positive individuals had a dominant Th2-type salivary cytokine profile (interleukin-4 [IL-4], IL-10) (IL-2, interferon-y [IFN-gamma], IL-12) that seemingly resulted from a lack of Th1-type cytokines rather than enhanced Th2-type cytokines. Moreover, pilot analyses of those with OPC showed evidence for a more profound salivary Th2-type profile. Both HIV-positive and HIV-negative patients, irrespective of CD4 counts, had some level of positive in vitro systemic lymphocyte proliferative responses to C albicans antigens. These results suggest that the Th1/Th2 cytokine dichotomy in HIV disease is detectable in situ in oral secretions and may be a useful indicator of oral-associated CMI to better understand resistance/susceptibility of HIV-positive individuals to oral opportunistic infections, including OPC.

Identification and Characterization of the Putative Fusion Peptide of the Severe Acute Respiratory Syndrome-Associated Coronavirus Spike Protein

ABSTRACT Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARSWW-I and SARSWW-II) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARSWW-I peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a β-sheet structure. Likewise, only SARSWW-I induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARSWW-I, we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

Abstract Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002–2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40–70% at concentrations of 15–30μM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2–4μM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Retroviruses and Their Roles in Chronic Inflammatory Diseases and Autoimmunity

Major advances in understanding the complex processes that regulate the immune system have been achieved. Elucidating the causes of human diseases characterized by inappropriate activation of the immune system, however, remains an elusive goal. Chronic inflammatory diseases or autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), appear to involve multifactorial pathogenic mechanisms. A genetic basis for susceptibility is established by the observation that occurrence of autoimmune diseases is substantially increased among family members of patients and is often related to inheritance of various genes in the human major histocompatibility complex (MHC). For example, the incidence of SLE is elevated in individuals that express certain haplotypes of the D-regulated (DR) and class III (complement, C) regions of the MHC (Doherty et al., 1992; Reveille, 1992). Other factors are also involved since only a fraction of SLE patients have the common haplotypes, and the concordance rate for SLE in identical twins is only about 30% (Christian et al., 1975; Imamura et al., 1975; Arnett and Shulman, 1976; Block et al., 1976). Many autoimmune diseases afflict predomi-nantly women, suggesting that hormonal factors may contribute to disease development and progression (Alarcon, 1993; Carlsten and Tarkowski, 1993). Furthermore, Afro-Americans are afflicted with SLE approximately three times as frequently as Americans of European descent, but blacks living in Africa do not appear to develop the disease with the same prevalence as their European or American counterparts (Clark et al., 1993).

The Hydrophobic Internal Region of Bovine Prion Protein Shares Structural and Functional Properties with HIV Type 1 Fusion Peptide

The conserved fusion peptide at the N-terminus of HIV-1 envelope glycoprotein gp41 is involved in the virus-cell fusion reaction and in the cytopathic effects promoted by expression in single cells. The conserved bovine prion protein 121KHVAGAAAAGAVVGGLGGYMLGSAMSR147 transmembrane region (BPrP(tm)) contains a sequence rich in Gly residues [i.e., 130GAVVGGLGGYMLGSAMSR147 (BPrP(mi))] that shows homology with HIV-1 fusion peptide. As in the case of the latter peptide, analysis of the BPrP(mi) interfacial hydrophobicity confirms that this stretch bears an intrinsic tendency to partitioning from the aqueous phase into membranes. Experimental analyses of BPrP(mi)-lipid interactions suggest several similarities between this sequence and HIV-1 fusion peptide. Infrared spectroscopy reveals a conformational plasticity of the membrane-associated prion sequence comparable to that displayed by the viral sequence. The adoption of a mainly alpha-helical structure correlates with the formation of lytic pores. This helical structure can be converted into a beta-sheet conformation by addition of calcium, a process that is accompanied by vesicle membrane fusion. In contrast, transmembrane BPrP(tm) associates with membranes in a nonlytic, mainly helical structure but also containing some random coil. Upon addition of calcium, the random coils disappear while the helical conformation remains. In the absence of membranes both prion and HIV-1 sequences form amyloid-type fibers. It is proposed that during the pathological processes induced by secreted PrPSc and its proteolytic fragments, conformational polymorphism displayed by membrane-inserted BPrP(mi) may play a role at perturbing the general architecture of the membrane lipid bilayer and inducing protein-protein aggregation at membrane surfaces. These findings suggest the existence of common mechanisms underlying cytotoxicity by PrP and HIV-1 gp41.

VIRAL INDUCTION, TRANSMISSION AND APOPTOSIS AMONG CELLS INFECTED BY A HUMAN INTRACISTERNAL A-TYPE RETROVIRUS

Sjogrens Syndrome, a systemic autoimmune disease, is characterized by lymphocytic infiltration of the salivary or lacrimal glands, producing xerostomia or xerophthalmia. Although definitive proof of viral etiology has not been established, a cell line containing viral particles termed Human Intracisternal A-type Particles (HIAP) resulted from co-culture with patient lip biopsies. We stimulated these chronically infected cells with phorbol myristate acetate (PMA) in an effort to enhance production of viral particles for further characterization. We report that the virus present in the HIAP cell line can be induced to become lytic when subjected to PMA and that there is a difference in the effects of PMA on H9 and HIAP cell groups, with apparent protection from apoptosis due to PMA being exerted by viral presence. Delayed apoptosis may prolong exposure of the foreign/self complex, thus enhancing an autoimmune response. Polyacrylamide gel electrophoresis (PAGE) revealed the presence of new peptides in pellets of supernatants of PMA-stimulated HIAP cells, with prominent bands at 55 and 43 kDa, and several fainter ones. HIAP infection was transferred by cell-free filtered supernatants from stimulated cells to H9 cells, which became identical to parent HIAP cells by PAGE and fluorescence activated cell sorter.

A quantitative assay for cytolysis induced by Newcastle disease virus.

We report here an assay for quantifying virus-induced lysis, in the absence of antibody and complement, produced within 2 hr after adsorption. This technique makes use of 51CrO4 release from cell monolayers pre-incubated overnight with the isotope. The release of 51Cr is specific for virus-induced lysis and is suppressible by 0.001 M-Ca2+. This assay clearly distinguishes between wild-type Chinese hamster ovary (CHO) cells, clone K and fusion-resistant mutant (CHO-15B), which was found to be resistant to virus-induced cytolysis. The stability of the association of isotope with monolayers of this cell type under the labelling conditions described makes this technique applicable to the study of the cytolytic effects of virus infection.

Production and characterization of a monoclonal antibody to dog hepatic lipase

Partially purified dog hepatic lipase was used as antigen to produce monoclonal antibodies in mice. In addition to enzyme-linked immunosorbent assay (ELISA), a reliable and efficient procedure for screening antibodies reacting to hepatic lipase has been developed. A method to distinguish antibodies directing to active site or non-active site epitopes has also been described. We obtained three positive clones that survived after subcloning and expansion. All three monoclonal antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. Specificity of monoclonal antibody LDHL No. 537 to dog hepatic lipase was demonstrated by passing post-heparin plasma through its immunoaffinity column. Only dog hepatic lipase was removed by LDHL No. 537 from post-heparin plasma. The immunoaffinity chromatography also demonstrated the co-existence of three enzyme activities (mono- and triacylglycerol lipase and phospholipase A1) on the dog hepatic lipase molecule. The subunit weight of dog hepatic lipase has been estimated at 57500 +/- 600 (n=3) by using immunoaffinity chromatography and the combination of immunoprecipitation and autoradiography methods.

Molecular character of influenza A/H1N1 2009: Implications for spread and control

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior “seasonal” H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 “swine flu” vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.