Spectroscopic study of an HIV‐1 capsid protein major homology region peptide analog

Abstract

The capsid (CA) domain of retroviral Gag proteins posseses one subdomain, the major homology region (MHR), which is conserved among nearly all avian and mammalian retroviruses. While it is known that the mutagenesis of residues in the MHR will impair virus infectivity, the precise structure and function of the MHR is not known. In order to obtain further information on the MHR, we have examined the structure of a synthetic peptide encompassing the MHR of human immunodeficiency virus type I (HIV-l) CA protein. Multiple sequence alignment and secondary structure prediction indicate that the peptide could form 50% o-helix and 10% P-sheet. In addition, circular dichroism studies indicate that, in the presence of 50% trifluoroethanol (TFE), the peptide adopts an cy-helical structure over half of its length. Further analysis by proton nuclear magnetic resonance spectroscopy suggests that the C-terminal portion of the MHR forms a helix in aqueous solution. Upon the addition of TFE, the position of the helix remains nearly constant, but the magnitude of the changes in H, chemical shifts of the residues indicate a more stable helix. These results suggest that a helical C-terminus of retroviral MHRs may be integral to the function of this region.

Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents

“…Insofar as CA cysteine substitution mutations are concerned, three were created in the C-terminal portion of HIV-1 MHR, since this region has been modeled to form an amphipathic helix (5), and one mutation was created at HIV gag codon 242, near a site which, when mutated by linker insertion, had a slight effect on assembly and processing (39). All of these mutants had phenotypes similar to that of the C330S mutant, although T242C was three times more infectious than C330S, and the MHR mutants were considerably less infectious (Table 1).…”

“…Previously, others found that mutations in the most conserved, amino-terminal residues of the MHR killed virus infectivity and could block assembly (27,37). We chose residues 294, 297, and 301 since nuclear magnetic resonance studies suggested that the HIV MHR forms an amphiphatic helix in solution with R-294 on the hydrophilic face and V-297 and Y-301 on the hydrophobic face (5). We felt that it was possible to determine whether cysteines introduced at these positions might interfere with assembly or, if not, might be used to probe structural features of the MHR.…”

Structural analysis of membrane-bound retrovirus capsid proteins

“…While MA interacts with viral membranes and Env proteins, and NC interacts with the viral RNA, the Gag CA domains appear to establish interprotein contacts that are essential to the oligomerization of Pr gag proteins, and assembly of Gag–Pol and Gag fusion proteins into virions (Hansen et al ., 1990; Jones et al ., 1990; Chazal et al ., 1994; Mammano et al ., 1994; Wang et al ., 1994; Craven et al ., 1995; Hansen and Barklis, 1995; Srinivasakumar et al ., 1995; McDermott et al ., 1996). Despite the apparent conservation of retrovirus capsid protein function, only a 20–30 residue section in the C‐terminal half of CA, called the major homology region (MHR), is well conserved at the primary sequence level (Mammano et al ., 1994; Craven et al ., 1995; Hansen and Barklis, 1995; Clish et al ., 1996; McDermott et al ., 1996). In addition to the three conserved mature Gag proteins (MA, CA and NC), M‐MuLV encodes a p12 protein between the MA and CA domains, and HIV encodes a p6 domain at the C‐terminus of its Pr gag : while these domains are essential to the infectivities of their respective viruses, they are not absolutely required for virus particle assembly (Crawford and Goff, 1984; Spearman et al ., 1994; Hansen and Barklis, 1995).…”

“…Three‐dimensional (3D) structures of HIV MA (Matthews et al ., 1994; Hill et al ., 1996) and NC (Morrelet et al ., 1992) are available, as are partial structures of HIV CA (Clish et al ., 1996; Gitti et al ., 1996; Momany et al ., 1996). However, despite observations of preferred interprotein contacts in 3D crystals (Hill et al ., 1996), it is unclear how the individual Gag proteins fit into the immature or mature virus particle structures.…”

Solution structures of human immunodeficiency virus type 1 (HIV‐1) and moloney murine leukemia virus (MoMLV) capsid protein major‐homology‐region peptide analogs by NMR spectroscopy

“…2), comprising CA residues 149Ϫ178 [23]. CD and NMR data indicated that this peptide assumes an A-helical conformation over half of its length in aqueous solution of 50% trifluoroethanol, while helical content could be detected through NMR chemical shift index (CSI) analysis in the absence of trifluoroethanol.…”