Potential role for CA-SP in nucleating retroviral capsid maturation.
During virion maturation, the Rous sarcoma virus (RSV) capsid protein is cleaved from the Gag protein as the proteolytic intermediate CA-SP. Further trimming at two C-terminal sites removes the spacer peptide (SP), producing the mature capsid proteins CA and CA-S. Abundant genetic and structural evidence shows that the SP plays a critical role in stabilizing hexameric Gag interactions that form immature particles. Freeing of CA-SP from Gag breaks immature interfaces and initiates the formation of mature capsids. The transient persistence of CA-SP in maturing virions and the identification of second-site mutations in SP that restore infectivity to maturation-defective mutant viruses led us to hypothesize that SP may play an important role in promoting the assembly of mature capsids. This study presents a biophysical and biochemical characterization of CA-SP and its assembly behavior. Our results confirm cryo-electron microscopy (cryo-EM) structures reported previously by Keller et al. (J. Virol. 87:13655-13664, 2013, doi:10.1128/JVI.01408-13) showing that monomeric CA-SP is fully capable of assembling into capsid-like structures identical to those formed by CA. Furthermore, SP confers aggressive assembly kinetics, which is suggestive of higher-affinity CA-SP interactions than observed with either of the mature capsid proteins. This aggressive assembly is largely independent of the SP amino acid sequence, but the formation of well-ordered particles is sensitive to the presence of the N-terminal β-hairpin. Additionally, CA-SP can nucleate the assembly of CA and CA-S. These results suggest a model in which CA-SP, once separated from the Gag lattice, can actively promote the interactions that form mature capsids and provide a nucleation point for mature capsid assembly.


OBJECTIVE
The spacer peptide is a documented target for antiretroviral therapy. This study examines the biochemical and biophysical properties of CA-SP, an intermediate form of the retrovirus capsid protein. The results demonstrate a previously unrecognized activity of SP in promoting capsid assembly during maturation.
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
Matthew R
MR
England
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
John G
JG
Purdy
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
Ira J
IJ
Ropson
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
Paula M
PM
Dalessio
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA rcraven@psu.edu.
Rebecca C
RC
Craven
D000595:Amino Acid Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020285:Cryoelectron Microscopy; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D052736:Rous sarcoma virus / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D019065:Virus Assembly
D036022:Capsid Proteins; D010446:Peptide Fragments