To define the role of the huma immuaodeciency virus type 1 (HIV-1) envelope proteins in virus infection, a series ofpeptides were synthesized based on various regions ofthe HIV-1 transmembrane protein gp4l. One of these peptides, DP-178, corresponding to a region predictive of a-helical secondary structure (residues 643-678 ofthe H1V-1LA isolate),.has been identified as a potent antivia agent. This peptide consistently blocked 100% of virus-mediated cell-cell fusion at <5 ng/ml (I(C2o 1.5 ng/ml) and gave an -10 times reduction in infectious titer of cel-free virus at =80 ng/ml. The inhibitory activity was observed at peptide concentrations _104 to 105 times lower than those at which cytotoxicity and cytostasis were detected. Peptidemediated inhibition is H1V-1 specific in that =102 to 10 times more peptide was required for inhibition of a human immunodeficiency virus type 2 isolate. Further experiments showed that DP-178 exhibited antiviral activity against both prototypic and primary IHV-1 isolates. As shown by PCR analysis of newly synthesized proviral DNA, DP-178 blocks an early step in the virus life cycle prior to reverse transcription. Finally, we discuss possible mechanisms by which DP-178 may exert its inhibitory activity.
A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.
Despite the presence of HIV-1 in the oral cavity, transmission of the virus through saliva has not been proven. Consistent with these observations, we recently identified an endogenous 12 kD protein, secretory leukocyte protease inhibitor (SLPI), in saliva which blocks HIV-1 infection in vitro. Whereas other salivary proteins tested were inactive, purified native or recombinant SLPI inhibited HIV-1 infection of human monocytes at 100 ng ml-1. Levels of SLPI quantitated by ELISA in saliva from control and HIV-1 infected individuals exceeded this level, consistent with in vivo antiviral activity. As in saliva, levels of SLPI mRNA determined by Northern hybridization, and protein as assessed by immunohistochemistry in the salivary glands of control and infected populations were comparable. In contrast to adults, oral transmission occurs in infants, possibly due to their lack of fully developed salivary glands. To determine whether the inadequate antiviral protection might be compensated for by maternal sources, we evaluated breast milk samples obtained 6 months postpartum. Levels of SLPI were significantly lower than in saliva and not sufficient to provide antiviral protection in contrast to colostrum samples in which SLPI levels were equivalent to those in saliva and able to inhibit HIV-1 infection when tested in vitro. These data suggest that breast milk may provide transient antiviral activity in the newborn, but that this maternal source of SLPI is of insufficient duration to maintain protection against mucosal transmission of the virus over time. The high functional levels of SLPI in saliva and the low levels in mature breast milk correlate with negligible rates of HIV-1 transmission by saliva and higher rates by breast feeding.
The nefgenes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (PXX)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.
Peak level HIV replication is established in blood, oropharyngeal tissues and genital tract, but potentially not in CSF, by the time patients are commonly diagnosed with primary HIV infection. Antiretroviral therapy is unlikely to limit initial virus spread to most tissue compartments, but may control genital tract shedding and central nervous system expansion in primary infection.
The oral cavity represents a unique site for mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Unlike other mucosal sites, the oral cavity is rarely a site of HIV transmission despite detectable virus in saliva and oropharyngeal tissues of infected persons. One reason for this apparent paradox is the presence of endogenous mucosal antiviral factors. Innate inhibitory molecules, such as virus-specific antibodies, mucins, thrombospondin, and soluble proteins, have been identified and partially characterized from saliva. A recent addition to the growing list is secretory leukocyte protease inhibitor (SLPI), an approximately 12-kDa non-glycosylated protein found in serous secretions. Physiologic concentrations of SLPI potently protect adherent monocytes and activated peripheral blood mononuclear cells against HIV-1 infection. SLPI levels in saliva and semen but not breast milk approximate levels required for inhibition in vitro. Characterization of SLPI and other endogenous antiviral molecules may enhance our understanding of factors influencing mucosal HIV-1 transmission.
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