Current HIV Research, 2007, 5, 69-78 69 1570-162X/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. Alteration of the Proline at Position 7 of the HIV-1 Spacer Peptide p1 Suppresses Viral Infectivity in a Strain Dependent Manner Melissa K. Hill *,1,2 , Anna Bellamy-McIntyre 1,2 , Laura J. Vella 1,§ , Shahan M. Campbell 1,¥ , John A. Marshall 3 , Gilda Tachedjian 1,2,4 and Johnson Mak 1,2,5 1 Virology Program, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia; 2 Department of Microbiology, Monash University, Clayton, Victoria, Australia; 3 Victorian Infectious Diseases Reference Laboratory (VIDRL), North Melbourne, Australia; 4 Department of Medicine, Monash University, Prahran, Victoria, Australia; 5 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia Abstract: The HIV-1 spacer peptide p1 is located in the C-terminus of the Gag polyprotein and separates the nucleocap- sid (NC) and p6 Gag . Research centered on p1 has been limited and as yet no function has been ascribed to this spacer pep- tide. We have previously found that the conserved p1 proline residues (position 7 and 13) are critical for replication in the HIV-1 strain HXB2-BH10. In this study we have focused on the proline rich p1-p6 Gag C-terminus of HIV-1. We individu- ally examined the role of p1 proline’s in multiple strains of HIV-1 and investigated the role of three proline residues in p6 Gag (P24, P25 and P30). Assessment of the HXB2-BH10 based mutants revealed that Gag-Pol incorporation relative to Gag decreased in the p1 mutant virions, with the double proline mutant the most impaired. Mutating both p1 proline resi- dues was found to abolish infectivity in multiple strains of HIV-1. Independent mutation of the p1 proline at position 7 re- sulted in a strain-dependent suppression of viral infectivity. This defect correlates with the presence of a tyrosine residue at position 9 of p1 and occurs in the early phase of the HIV-1 replication cycle. The p1 proline residues were found to be functionally distinct from P24, P25 and P30 in p6 Gag . This work affords novel insights into our understanding of the role of p1 in HIV-1 replication. Keywords: HIV, spacer peptide, p1, p6 Gag , Gag. INTRODUCTION The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) is the fundamental driving force behind viral assembly, encoding the viral structural proteins and harbour- ing key assembly domains. Cleavage of Gag by the viral protease (PR) releases the mature proteins matrix (MA), cap- sid (CA) nucleocapsid (NC) and p6 Gag . Two small spacer peptides, p2 and p1, are also found within HIV-1 Gag. The 14 amino acid p2 spacer peptide separates CA and NC and the 16 amino acid p1 spacer peptide separates NC and p6 Gag (Fig. 1A). Numerous studies have explored the role of the p2 spacer peptide in HIV-1 replication, demonstrating that p2 tran- siently supports: 1) the proteolytic processing of viral pre- cursor proteins; 2) assembly of virion particles; and 3) the selective virion packaging of HIV-1 RNA genomes [1, 18, 19, 25, 27, 28, 29, 34, 35]. Moreover, it has been shown that the cleavage of p2 acts as a regulatory switch for the mor- phological conversion of newly assembled immature virions *Address correspondence to this author at the Macfarlane Burnet Institute for Medical Research and Public Health. G.P.O. Box 2284, Melbourne, Victoria, Australia, 3001; Tel: 61 3 9282 2124; Fax: 61 3 9282 2100; E- mail: mel@burnet.edu.au § Present address: Department of Pathology, Centre for Neuroscience, and Mental Health Research Institute of Victoria, University of Melbourne, Parkville, Victoria, Australia ¥ Present address: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia to mature HIV-1 particles [18]. The function of p2 is reliant on protein structure, wherein the formation of an alpha-helix that spans the CA-p2-NC junction influences the folding of CA and NC to increase the helix forming tendency of both proteins during Gag/Gag interactions and Gag/HIV-RNA interactions [28]. Accordingly, changes to p2 structure di- rectly inhibit the production of infectious HIV-1 [1]. Comparatively, investigations into the function of p1 have been rare. The p1 spacer peptide is located at the point where the critical RNA structure of the frameshift stem-loop overlaps with both the Gag p1 and the Gag-Pol transframe (TF) open reading frames. Consequently, mutagenesis of this region has the potential to interfere with the functioning of multiple elements. A systematic mutagenesis strategy to iso- late p1 function enabled us to demonstrate a critical role for p1 in HIV-1 replication that is clearly independent of the roles of the RNA frameshift stem-loop and the Gag-Pol TF protein [20]. We further determined that simultaneous altera- tion of p1’s two conserved proline residues (position 7 and 13) to leucines influenced protein processing, reduced ge- nomic RNA dimer stability and abolished viral infectivity in peripheral blood mononuclear cells (PBMCs) [20]. The nature of the involvement of p1 in HIV-1 function remains unclear. As proline residues can confer unique con- formational constraints on a peptide, our results suggest that like the analogous p2, the structure of p1 is critical. The im- pact of p1 on HIV-1 biology is anticipated to be derived from a contribution to the overall protein folding of one of the precursor proteins that encompass p1, such as Gag (MA-