Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

Abstract

BackgroundThe equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.ResultsIn the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.ConclusionsThese structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

“…First, we analyzed the equine infectious anemia virus (EIAV) p9 Gag protein, which represents the L-domain containing EIAV homolog of HIV-1 p6. With a size (51 aa) comparable to HIV-1 p6, it displays little sequence homology to HIV-1 p6 (Fig 4G) [28,29]. In contrast to s p6, there was no degradation observed for s p9 under conditions in which s p6 was rapidly degraded (Fig 4A and 4B).…”

“…(G) Sequence alignment of p6 peptides from HIV-1, HIV-2, SIV and the EIAV p9 peptide. The sequence of HIV-2 p6 originates from the isolate ROD10, SIV p6 from SIVmac239, and EIAV p9 from the isolate EIAV Wyoming [29]. …”

“…Peptide sequence for synthetic HIV-2 p6 was derived from the isolate ROD10 and SIV p6 originates from SIVmac239. Synthesis of unlabeled s p6 wt and s p9 has been described earlier [18,29]. The sequence of the p6 mutant E0A has been published previously [24].…”

The N-Terminus of the HIV-1 p6 Gag Protein Regulates Susceptibility to Degradation by IDE

“…( C ) Sequence alignment of IDE insensitive p6 peptides from HIV-2, SIV and EIAV p9 peptide [16]. The sequence of HIV-2 p6 originates from the isolate ROD10, SIV p6 from SIVmac239, and EIAV p9 from the isolate EIAV Wyoming [16,58]. Colors of the sequence alignments according to the physicochemical properties of the aa, as proposed by the Clustal Omega multiple sequence alignment software [45,46,47].…”