Second-Site Suppressors of Rous Sarcoma Virus CA Mutations: Evidence for Interdomain Interactions

Abstract

The capsid (CA) protein, the major structural component of retroviruses, forms a shell that encases the ribonucleoprotein complex in the virion core. The most conserved region of CA, ϳ20 amino acids of the major homology region (MHR), lies within the carboxy-terminal domain of the protein. Structural and sequence similarities among CA proteins of retroviruses and the CA-like proteins of hepatitis B virus and various retrotransposons suggest that the MHR is involved in an aspect of replication common to these reversetranscribing elements. Conservative substitutions in this region of the Rous sarcoma virus protein were lethal due to a severe deficiency in reverse transcription, in spite of the presence of an intact genome and active reverse transcriptase in the particles. This finding suggests that the mutations interfered with normal interactions among these constituents. A total of four genetic suppressors of three lethal MHR mutations have now been identified. All four map to the sequence encoding the CA-spacer peptide (SP) region of Gag. The F167Y mutation in the MHR was fully suppressed by a single amino acid change in the alpha helix immediately downstream of the MHR, a region that forms the major dimer interface in human immunodeficiency virus CA. This finding suggests that the F167Y mutation indirectly interfered with dimerization. The F167Y defect could also be repaired by a second, independent suppressor in the C-terminal SP that was removed from CA during maturation. This single residue change, which increased the rate of SP cleavage, apparently corrected the F167Y defect by modifying the maturation pathway. More surprising was the isolation of suppressors of the R170Q and L171V MHR mutations, which mapped to the N-terminal domain of the CA protein. This finding suggests that the two domains, which in the monomeric protein are separated by a flexible linker, must communicate with each other at some unidentified point in the viral replication cycle.

Higher-Order Structure of the Rous Sarcoma Virus SP Assembly Domain

“…In fact, using the maturation inhibitor PF-46396, Waki et al mapped a novel binding pocket formed by the major homology region (MHR) of HIV-1 CA and SP1 and predicted that these two distant regions are in close proximity in an assembled virus (89). This inference is supported by data from Bowzard et al that showed the rescue of a lethal MHR mutation in RSV CA by a second-site suppressor mutation in SP (90). Given the apparent conservation of an SP-like domain and the absolute conservation of the MHR across all retroviral genera, perhaps this putative interaction surface between SP and the MHR should be further investigated for its role in stabilizing the immature retrovirus.…”

The structural biology of HIV assembly

“…Gratifyingly, this pseudoatomic model of the hexagonal capsid lattice is consistent with a large body of biochemical and genetic data, including deuterium exchange, crosslinking, mutagenesis, and second-site suppressor studies (e.g., see Fig. 3c and d) [47,58,98,103,104]. Although a molecular model for the hexagonal CA lattice is now in hand, we still lack a comprehensive, high-resolution structural model of the mature HIV-1 capsid.…”

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

“…Similar studies have recently been performed on the CA protein of RSV (3,7). Several mutations in the major homology region of RSV resulted in efficient assembly of virions that were blocked for reverse transcription in target cells and exhibited core stability defects (7).…”