Many retroviruses, including the human and simian immunodeficiency viruses, contain a leucine zipper-like repeat in a highly conserved region of the external domain of the transmembrane (TM) glycoprotein. This region has been postulated to play a role in stabilizing the oligomeric form of these molecules. To determine what role this region might play in envelope structure and function, several mutations were engineered into the middle isoleucine of the leucine zipper-like repeat of the human immunodeficiency virus type 1 (HIV-1) TM protein. A phenotypic analysis of these mutants demonstrated that conservative mutations (Ile to Val or Leu) did not block the ability of the viral glycoprotein to mediate cell-cell fusion or affect virus infectivity. In contrast, each of the other mutations, except for the Ile-to-Ala change, completely inhibited the ability of the glycoprotein to fuse HeLa-T4 cells and of mutant virions to infect H9 cells. The alanine mutation produced an intermediate phenotype in which both cell fusion and infectivity were significantly reduced. Thus, the biological activity of the glycoprotein titrates with the hydrophobicity of the residue in this position. None of the mutations affected the synthesis, oligomer formation, transport, or processing of the HIV glycoprotein complex. Although these results do not rule out a role for the leucine zipper region in glycoprotein oligomerization, they clearly point to a critical role for it in a post-CD4 binding step in HIV membrane fusion and virus entry.
The envelope glycoprotein precursor of retroviruses undergoes proteolytic cleavage in the Golgi complex to yield the mature surface and transmembrane (TM) glycoproteins of the virus. We report here that the TM glycoprotein of Mason-Pfizer monkey virus undergoes a second proteolytic processing event during a late maturation step that can follow virus release and Gag polyprotein cleavage. Cleavage results in the conversion of the cell-associated TM glycoprotein (gp22) to a virus-associated gp2O. Processing continues after virus release and yields virions that contain predominantly gp2O. A mutation within the active site of the Mason-Pfizer monkey virus aspartyl protease was shown to block both TM glycoprotein cleavage and the processing of the Gag polyprotein precursor. The role of the viral protease in cleavage of the TM glycoprotein localizes the cleavage site to the cytoplasmic domain of this protein. Surprisingly, point mutations within the matrix (MA) coding region of the gag gene can affect the extent to which gp22 is processed to gp2O and in one case
The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr843 and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr843 or Tyr863 is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of α-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab′)2 blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.
Viral protease-mediated cleavage within the cytoplasmic domain of the transmembrane (TM) glycoprotein of the type D retrovirus, Mason-Pfizer monkey virus, removes approximately 16 amino acids from the carboxy terminus of the protein. To determine the functional significance of this cleavage in the virus life cycle, we introduced premature stop codons into the TM coding domain, resulting in the production of truncated glycoproteins. Progressive truncation of the cytoplasmic domain identified the carboxy-terminal third as being required for efficient incorporation of the glycoprotein complex into budding virions and profoundly increased the fusogenic capability of the TM glycoprotein. These results, together with the ability of matrix protein mutations to suppress TM cleavage, imply that this portion of the glycoprotein interacts specifically with the capsid proteins during budding, suppressing glycoprotein fusion function until virus maturation has occurred.
Healthy individuals at high risk of exposure to ticks were surveyed to determine the prevalence and incidence of asymptomatic babesiosis on Shelter Island, New York, during a single transmission season. Paired sera obtained in June and October 1978 were tested for antibodies to Babesia microti by indirect immunofluorescence. Point prevalence values of 4.4% in June and 6.9% in October were obtained. Six of 102 persons tested in both months showed at least a fourfold rise in titer of antibodies to B. microti (an incidence of 5.9% for the season). None of 300 serum specimens obtained from the New York City metropolitan area had significant titers to B. microti. Of the six persons who seroconverted, four gave a history of tick bite during the transmission season; three of the four persons claimed to recognize the tick as an Ixodes dammini, the vector for B. microti.
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