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Mannose-binding lectin (MBL), an acute-phase serum protein of hepatic origin, plays an essential protective role in host innate immunity in targeting microbial pathogens for destruction via opsonization, enhancement of phagocytosis and complement activation. MBL deficiency, characterized by low serum MBL, has been attributed to genetic mutations in both structural and promoter regions of the gene coding for the protein. Concomitant MBL deficiency in patients with chronic immunologic disease has been associated with increased susceptibility to complicating infections that may hasten disease progression.

ABSTRACTCyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.

ABSTRACTA cytopathic variant of feline immunodeficiency virus (FIV) strain PPR emerged after passage of wild-type virus on an interleukin-2-independent cell line. The virus, termed FIV-PPRglial, displayed a phenotype markedly different from the parental virus, including the ability to productively infect previously refractory cell lines, induction of large syncytia, and accelerated kinetic properties. A chimeric molecular clone, FIV-PPRchim42, containing the FIV-PPRglial envelope within the backbone of FIV-PPR, exhibited all the characteristics of the FIV-PPRglial phenotype, demonstrating that the viral envelope was responsible for the acquired traits. Subsequent molecular characterization revealed that the FIV-PPRglial envelope contained five amino acid substitutions relative to wild-type FIV-PPR. Mutagenic analyses further demonstrated that the acquired phenotype was minimally attributable to a combination of three mutations, specifically, a glutamine-to-proline change within the second constant domain of the surface protein (SU); a threonine-to-proline change within the V4 loop, also in the SU; and a premature stop codon in the cytoplasmic tail of the transmembrane protein. All three changes were required to produce the FIV-PPRglial phenotype. Cotransfection studies with mutant viruses in combination with each other and with FIV-PPR indicated that the truncated cytoplasmic tail was responsible for the induction of syncytium formation. Receptor usage analyses were pursued, and distinctions were observed between FIV-PPR and FIV-PPRglial. In vitro infections with FIV-PPR, FIV-PPRglial, and FIV-34TF10 on two adherent cell lines were ablated in the presence of SDF1a, the natural ligand for CXCR4. In contrast, viral infection of T cells was not limited to CXCR4 usage, and inhibition studies indicate the potential involvement of a CC chemokine receptor.

The feline lentivirus, FIV, contains dUTPase (DU) as part of the enzyme cassette encoded by the viral pol gene (Elder et al., 1992, J. Virol. 66, 1791-1794). The enzyme is processed from the Pol polyprotein and is packaged into infectious virions. We report here the basic characteristics of the viral enzyme, including substrate specificity, ion requirements, and pH optimum. We also report the overexpression of DU in Escherichia coli and insertional mutagenesis of the enzyme in the context of the complete provirus or DU alone. The enzyme requires Mg²⁺ for full activity and competition studies employing unlabeled dNTPs indicated that DU has an absolute preference for dUTP. The pH optimum for FIV DU is pH 7.0. The limits of the protein dictate a species of M_r 14,350, which agrees precisely with the determination by ion spray mass spectroscopy of DU isolated from virions. Cleavage sites at the junctions between DU, RT, and IN, as defined by N-terminal amino acid sequencing of each protein species, are consistent with predictions for sites of cleavage by aspartate protease. In-frame insertional mutagenesis at Tyr 75 of DU abolishes activity. Cells transfected with proviruses containing this mutation express virion-associated reverse transcriptase activity but lack DU activity. The resultant virions replicate slower than those possessing wild-type DU. Tests are currently underway to evaluate the consequences of DU mutagenesis on in vivo phenotype. Copyright 1993, 1999 Academic Press