Abstract:The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a d… Show more
“…Given that viruses lacking the V1/V2 region or the V2 variable loop are capable of replication 74,75 , this region cannot be essential for infectivity and therefore is relatively unconstrained in terms of mutation. Therefore, despite the fact that this region of the virus envelope carries out important functions -mediating gp41-independent interaction within the gp120 trimer 76 and shielding the region in and around the bridging sheet of gp120 responsible for interaction with chemokine receptors 74,75,77-79 -the V1/V2 loop is an improbable target for vaccines and, in fact, is specifically being deleted in some vaccine constructs to provide better access to neutralizing epitopes in and near the co-receptor binding site 80 .…”
Section: Variable Regions Of the Virus Envelope As Targetsmentioning
“…Given that viruses lacking the V1/V2 region or the V2 variable loop are capable of replication 74,75 , this region cannot be essential for infectivity and therefore is relatively unconstrained in terms of mutation. Therefore, despite the fact that this region of the virus envelope carries out important functions -mediating gp41-independent interaction within the gp120 trimer 76 and shielding the region in and around the bridging sheet of gp120 responsible for interaction with chemokine receptors 74,75,77-79 -the V1/V2 loop is an improbable target for vaccines and, in fact, is specifically being deleted in some vaccine constructs to provide better access to neutralizing epitopes in and near the co-receptor binding site 80 .…”
Section: Variable Regions Of the Virus Envelope As Targetsmentioning
“…Methods for the estimation of average partial specific volumes have been described by Shire (1992), Junghans et al (1996), Fairman et al (1999), and Lewis and Junghans (2000). For glycosylated proteins, the availability of molar mass data from mass spectrometry is extremely helpful, because, together with the molar mass of the protein component as calculated from amino acid composition, the molar mass of the carbohydrate component can be estimated (Fairman et al, 1999;Center et al, 2000). A technique in which two SE experiments are performed simultaneously, one in D 2 O and one in H 2 O (Edelstein and Schachman, 1967), can be useful in the study of glycosylated proteins.…”
Section: Size Partial-specific Volume and Prior Characterization Ofmentioning
This unit describes basic principles and practice of sedimentation equilibrium analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, and binding stoichiometry, as well as the determination of association constants. Advanced tools such as mass conservation analysis, multiwavelength analysis, and global analysis are introduced and discussed in the context of the experimental design. A detailed protocol guiding the investigator through the experimental steps and the data analysis is available as an internet resource.
“…Major findings of this work are that (i) the SU subunit of the SARS-CoV S glycoprotein (S1) forms dimers, (ii) the dimerization domain does not overlap and is upstream of the RBD, (iii) its deletion abolishes fusion, (iv) dimeric S1 binds receptor molecules much more efficiently than monovalent fragments containing the RBD, and (v) the soluble S ectodomain forms trimers under gel filtration conditions. It has been previously reported that some attachment SU subunits of class I fusion proteins that bind receptor molecules can form dimers including gp120 of the retrovirus HIV-1 [17] and S1 of the coronavirus MHV [16]. What is the role of S1 dimerization for mediation of membrane fusion however remains unclear.…”
Viral envelope glycoproteins are oligomeric and the quaternary structure is critical for their membrane fusion activity. Typically the transmembrane glycoproteins of class I fusion proteins contain the oligomerization domains and the surface glycoproteins (SU) are monomeric. However, it has been previously demonstrated [J. Biol. Chem. 277 (2002) 19727] that the SU of a murine hepatitis coronavirus (MHV) forms dimers, the dimerization domain overlaps the receptor-binding domain (RBD) and that this dimeric state is important for binding to receptor molecules that initiates entry into cells. We have previously expressed various soluble fragments of the SARS-CoV SU and identified stably folded fragments (residues 272-537) that contain the RBD [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Here, we further characterize these and other fragments in an attempt to identify possible dimerization domains and their role for membrane fusion. We demonstrate that the SU and a shorter 260-amino acid N-terminal fragment (residues 17-276), which folds independently, form dimers. In contrast to the previously characterized MHV SU dimerization, this fragment is upstream and distinct from the RBD. Its deletion abolished S-mediated cell membrane fusion but retained the SU-receptor-binding function indicating the possibility for a role in post-receptor binding steps of the virus entry mechanism. Interestingly, the whole soluble S ectodomain (Se) that contains the dimerization domain but not the transmembrane domain and the cytoplasmic tail forms trimers suggesting the existence of a trimerization domain in the TM subunit in its prefusion state that may lead to a conformation unfavorable for formation of higher-order multimeric structures. These results demonstrate the existence of SU dimers and Se trimers, and indicate the possibility for an unknown mechanism of their role in entry. They also further characterize the S-mediated membrane fusion and could be important for understanding the mechanisms of virus entry, and in the development of therapeutics and vaccines.
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