The ubiquitous eukaryotic protein calreticulin has been detected in a wide variety of different cell types. Recently, calreticulin was found to bind in vitro to a number of proteins isolated from the endoplasmic reticulum. In addition, calreticulin has sequence similarities with the molecular chaperone calnexin. These data suggest that calreticulin might also act as a chaperone. We found that calreticulin associated transiently with a large number of newly synthesized cellular proteins. In cells expressing recombinant human immunodeficiency virus (HIV) envelope glycoprotein, gp160 bound transiently to calreticulin with a peak at 10 min after its synthesis. Binding of gp120 to calreticulin was not detected because proteolytic cleavage of gp160 occurs in the trans-Golgi. Nonglycosylated HIV envelope protein was not associated with calreticulin, suggesting a requirement for N-linked oligosaccharides on newly synthesized proteins as has been reported for calnexin. The in vivo binding kinetics of calnexin and calreticulin to gp160 were very similar. Sequential immunoprecipitations provided evidence for the existence of ternary complexes of gp160, calreticulin, and calnexin. The data suggested that most of the gp160 associated with calreticulin was also bound to calnexin but that only a portion of the gp160 associated with calnexin was also bound to calreticulin.
Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min lag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1-to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.
Recently, combinations of antiretroviral drugs (highly active antiretroviral therapy [HAART]) have led to a dramatic reduction of human immunodeficiency virus type 1 (HIV-1)-related clinical symptoms. Success of treatment is defined as almost complete suppression of plasma viremia, although in a sizable fraction of patients this goal is not achieved. We characterized primary HIV-1 isolates from 2 cohorts of patients in which HAART failed in terms of viral suppression. One cohort showed clinical benefit and stable or increasing CD4+ T cell numbers despite high viral load. The second viremic cohort had no CD4+ T cell recovery and exhibited typical AIDS-related symptoms. Primary isolates from HAART patients with minor clinical symptoms used CXCR4 as the most relevant receptor on primary cells. Thus, for the first time, it is shown that patients improving clinically under HAART harbor relatively high viral loads with viruses preferring CXCR4 as coreceptor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.