Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6Gag
© Radestock et al.; licensee BioMed Central. 2014
Received: 23 September 2014
Accepted: 17 November 2014
Published: 19 December 2014
HIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6Gag had only minor effects.
We introduced mutations into p6Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.
An HIV-1 variant carrying a conservative S40N exchange in p6Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.
KeywordsHuman immunodeficiency virus Gag p6 Phosphorylation Release Serine 40
HIV-1 assembly is driven by the viral Gag polyprotein. Gag is necessary and sufficient for particle formation, and is composed of four functional subunits. The N-terminal MA domain targets Gag to the plasma membrane, where ~2,500 Gag molecules form the curved, hexagonal immature lattice of HIV-1, which is stabilized by intermolecular CA domain interactions. The NC domain is responsible for packaging viral genomic RNA . Crucial motifs for particle release lie within the 52 amino acids long C-terminal p6Gag domain. This domain harbors so-called late domains, which recruit the cellular endosomal sorting complex required for transport (ESCRT) promoting abscission of progeny particles from the host cell. Other motifs in p6Gag mediate incorporation of the viral accessory protein Vpr into HIV-1 particles -. During maturation, immature HIV-1 gains infectivity following proteolytic cleavage of Gag into its functional domains by the viral protease .
p6Gag has been shown to be the predominant phosphoprotein in HIV-1 particles . It is phosphorylated at several positions, including the highly conserved residue S40 -. S40 phosphorylation has been detected in infected cells and viral particles , and this residue can be phosphorylated by atypical protein kinase C (aPKC) in vitro .
To determine the role of p6 phosphorylation for HIV-1 replication, we had recently performed a comprehensive mutational analysis of p6 . The use of an HIV-1NL4-3 based proviral plasmid with genetically uncoupled gag and pol open reading frames (ORFs) (pNL4-3unc) allowed us to freely introduce mutations in the p6 gag encoding region without affecting the pol ORF. In this context, we changed all phosphorylatable residues (i.e., Ser, Thr, Tyr) within p6Gag with exception of the essential threonine in the PTAP late domain motif. The resulting virus, NL4-3uncFL exhibited no significant difference in replication capacity compared to wild-type. This result led us to conclude that p6 phosphorylation is dispensable for viral morphogenesis and replication in cell culture.
In contrast, previous studies had reported that an S40F change in p6Gag impaired proteolytic maturation of Gag, reduced viral infectivity and delayed replication in T-cell lines ,. Furthermore, enhanced membrane binding affinity of a synthetic p6 C-terminal fragment was observed in vitro upon substitution of Ser40 by Phe or upon adding a phosphate group to this residue . The S40F exchange was furthermore shown to result in an enhanced interaction of Gag with the ESCRT-associated protein Alix . Taken together, these studies suggested an important role of S40 in Gag assembly , viral maturation , Vpr incorporation , and p6 membrane binding , in apparent contradiction to our observation that an HIV-1 derivative carrying mutations at 12 positions within p6, including S40, was fully functional in cell culture .
A major difference between our work  and the studies reported by others ,- was that the latter employed a chemically drastic Ser to Phe exchange in order to maintain the amino acid sequence of the overlapping pol ORF, whereas the gag-pol uncoupling strategy allowed us to select the most conservative substitution, Ser to Asn. In order to resolve the apparent discrepancies between our study and data published by others, we performed a direct side-by-side comparison of viruses carrying either an Asn or a Phe residue at position 40 of p6Gag.
For assessment of virus release, culture media were harvested 30 h post transfection (p.t.), cleared by brief centrifugation followed by ultracentrifugation through a 20% (w/w) sucrose cushion to pellet virus particles. Samples of cell and particle lysates were separated by SDS-PAGE and proteins were transferred to a PVDF membrane. HIV-1 CA-containing proteins were detected by quantitative immunoblotting using polyclonal sheep antiserum against recombinant CA (Figure 1B), and HIV-1 particle release was quantified by determining the ratio of the amount of pelletable extracellular CA-containing proteins over the total amount of CA-containing proteins (Figure 1C). As expected, NL4-3 late(-) showed strongly reduced particle release compared to wild-type, accompanied by a characteristic increase in the proportion of processing intermediates, in particular CA-SP1. Variants NL4-3uncFL, FL-N40S, and S40N displayed wild-type Gag processing and particle release. Increased amounts of the CA-SP1 processing intermediate were observed in case of the S40F variant, consistent with previous reports ,, and particle release was also slightly higher in this case (Figure 1B,C).
In summary, our analyses confirm previous reports that an S40F substitution in p6Gag of HIV-1 impairs Gag processing at the CA-SP1 site and severely affects or abolishes HIV-1 replication in cell lines and primary cells ,. This effect is not due to either a requirement for the conserved serine residue at this position or for phosphorylation of S40. The conservative substitution of S40 by a chemically similar, but not phosphorylatable Asn residue had no effect on Gag processing or viral infectivity in cell lines or primary T-cells compared to wild-type HIV-1. Thus, the previously described replication defect of the S40F variant appears to be due to the replacement of the small, hydrophilic serine by a bulky hydrophobic phenylalanine residue, rather than indicating a requirement for S40. We did not analyze Gag membrane-binding properties, which had been reported to be affected by the S40F substitution or by introducing a phosphomimick in this position . However, wild-type release, polyprotein processing and infectivity of all variants studied here except for S40F suggest that neither S40 nor its phosphorylation is needed for fully functional membrane binding of Gag. Furthermore, we did not observe a block in Vpr incorporation upon mutation of S40, which had been reported for an S40A variant in a previous study and had been proposed to result in impaired replication in primary macrophages . While we cannot exclude that S40 in p6Gag and/or its phosphorylation may be relevant in a different cell context (e.g., macrophages), we conclude that the phenotypes reported in previously published studies ,- were most likely caused by the specific mutation introduced and do not reflect the functional importance of Ser40 or its phosphorylation.
We thank Bärbel Glass for technical assistance. The plasmid pNL43_E-_R-_Luc3/p6:M1A, which was used to produce pNL4-3 Vpr(-), was kindly provided by Nathaniel Landau (New York). This work was supported in part by the Deutsche Forschungsgemeinschaft through the collaborative research grant SFB638 (project A9). HGK and BM are investigators of the excellence cluster CellNetworks (EXC81). RB is supported by the Hartmut Hoffman-Berling International Graduate School of Molecular and Cellular Biology.
- Sundquist WI, Krausslich HG: HIV-1 Assembly, Budding, and Maturation. Cold Spring Harb Perspect Med. 2012, 2: a006924-10.1101/cshperspect.a015420.PubMed CentralView ArticlePubMedGoogle Scholar
- Kondo E, Gottlinger HG: A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr. J Virol. 1996, 70: 159-164.PubMed CentralPubMedGoogle Scholar
- Sunseri N, O'Brien M, Bhardwaj N, Landau NR: Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells. J Virol. 2011, 85: 6263-6274. 10.1128/JVI.00346-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Paxton W, Connor RI, Landau NR: Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993, 67: 7229-7237.PubMed CentralPubMedGoogle Scholar
- Muller B, Patschinsky T, Krausslich HG: The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles. J Virol. 2002, 76: 1015-1024. 10.1128/JVI.76.3.1015-1024.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Kudoh A, Takahama S, Sawasaki T, Ode H, Yokoyama M, Okayama A, Ishikawa A, Miyakawa K, Matsunaga S, Kimura H, Suqiura W, Sato H, Hirano H, Ohno S, Yamamoto N, Ryo A: The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions. Retrovirology. 2014, 11: 9-10.1186/1742-4690-11-9.PubMed CentralView ArticlePubMedGoogle Scholar
- Radestock B, Morales I, Rahman SA, Radau S, Glass B, Zahedi RP, Muller B, Krausslich HG: Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication. J Virol. 2013, 87: 724-734. 10.1128/JVI.02162-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Hemonnot B, Cartier C, Gay B, Rebuffat S, Bardy M, Devaux C, Boyer V, Briant L: The host cell MAP kinase ERK-2 regulates viral assembly and release by phosphorylating the p6gag protein of HIV-1. J Biol Chem. 2004, 279: 32426-32434. 10.1074/jbc.M313137200.View ArticlePubMedGoogle Scholar
- Watanabe SM, Chen MH, Khan M, Ehrlich L, Kemal KS, Weiser B, Shi B, Chen C, Powell M, Anastos K, Burger H, Carter CA: The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities. Retrovirology. 2013, 10: 143-10.1186/1742-4690-10-143.PubMed CentralView ArticlePubMedGoogle Scholar
- Votteler J, Neumann L, Hahn S, Hahn F, Rauch P, Schmidt K, Studtrucker N, Solbak SM, Fossen T, Henklein P, Ott DE, Holland G, Bannert N, Schubert U: Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly. Retrovirology. 2011, 8: 11-10.1186/1742-4690-8-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Solbak SM, Reksten TR, Hahn F, Wray V, Henklein P, Henklein P, Halskau O, Schubert U, Fossen T: HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein. Biochim Biophys Acta. 1828, 2013: 816-823.Google Scholar
- Gottlinger HG, Dorfman T, Sodroski JG, Haseltine WA: Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci U S A. 1991, 88: 3195-3199. 10.1073/pnas.88.8.3195.PubMed CentralView ArticlePubMedGoogle Scholar
- Sena-Esteves M, Saeki Y, Camp SM, Chiocca EA, Breakefield XO: Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons. J Virol. 1999, 73: 10426-10439.PubMed CentralPubMedGoogle Scholar
- Huang M, Orenstein JM, Martin MA, Freed EO: p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J Virol. 1995, 69: 6810-6818.PubMed CentralPubMedGoogle Scholar
- Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986, 59: 284-291.PubMed CentralPubMedGoogle Scholar
- Connor RI, Chen BK, Choe S, Landau NR: Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology. 1995, 206: 935-944. 10.1006/viro.1995.1016.View ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu X, Shaw GM, Kappes JC: Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother. 2002, 46: 1896-1905. 10.1128/AAC.46.6.1896-1905.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Salahuddin SZ, Markham PD, Wong-Staal F, Franchini G, Kalyanaraman VS, Gallo RC: Restricted expression of human T-cell leukemia–lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology. 1983, 129: 51-64. 10.1016/0042-6822(83)90395-1.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.