Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca 2؉ -binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(؉)MA) domain. Herein, we demonstrate that CaM binds to myr(؉)MA with a dissociation constant (K d ) of ϳ2 M and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(؉)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.Gag is the major structural protein encoded by HIV-1 and contains all of the viral elements required to drive virus assembly (1-3). HIV-1 Gag targeting to the plasma membrane (PM) 2 is critical for proper and efficient assembly to produce progeny virions (1, 3-9). During virus maturation, Gag is cleaved into myristoylated matrix (myr(ϩ)MA), capsid, and nucleocapsid proteins, inducing major morphological reorganization of the virus (1, 2, 4, 5, 10). In many cell types, HIV-1 Gag budding and assembly has been shown to occur predominantly on the PM (4 -9, 11-18). Gag binding to the PM is mediated by the MA domain and enhanced by multimerization. Proper assembly and efficient binding of Gag to the PM requires a myristyl (myr) group as a membrane anchor and a cluster of basic residues localized within the N-terminal domain to facilitate interactions with acidic phospholipids (1,2,19,20).Steady progress has been made in defining both the viral and cellular determinants of HIV-1 assembly and release (6). However, the trafficking pathway used by Gag to reach assembly sites in the infected cell is poorly understood. Studies by Freed, Ono, and co-workers (21-23) demonstrated that the ultimate localization of HIV-1 Gag at virus assembly sites is dependent on phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ), a cellular factor localized at the inner leaflet of the PM (24 -26). Our structural studies revealed that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myr exposure (27). In addition to PI(4,5)P 2 ...

Human immunodeficiency virus type-1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix (myr(+)MA) domain of Gag and phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ). Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P 2 binding and is enhanced by factors that promote protein self-association. In the studies reported herein, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His-Glu salt bridge by single amino acid substitutions greatly altered the myr-sequestered-myr-exposed equilibrium. In vivo intracellular localization data revealed that H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a "pH sensor" in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation. KeywordsHuman immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2); myristyl (myr); matrix (MA); nuclear magnetic resonance (NMR); His-Glu salt bridge Corresponding Author: Jamil S. Saad, Ph.D., 845 19 th Street South, Birmingham, AL 35294;; saad@uab.edu. SUPPORTING INFORMATION AVAILABLE 2D 1 H-15 N HSQC NMR spectra of myr(−) and myr(+)MA as a function of pH (Figs. S1 and S2); 3D 13 C-edited/ 12 C-double-halffiltered NOE spectrum of myr(+)MA at pH 5.5 (Fig. S3); sedimentation equilibrium profiles for myr(+)MA at different pH values (Fig. S4); plots of chemical shift changes vs. pH used to determine the pK a value of His89 for myr(−)MA (Fig. S5); sedimentation equilibrium and velocity profiles for WT CA and various MACA mutants as a function of pH ( Fig. S6-S9); 2D NMR HSQC spectra (Fig. S10), sedimentation velocity profiles ( Fig. S11) and CD spectra (Fig. S12) for MA H89G mutants; 2D HSQC spectra and sedimentation velocity profiles of MA E12A mutants (Fig. S13); sedimentation velocity profiles for myr(+)MA-E12A/W184A/ M185A as a function of pH (Fig. S14); structural representation of salt bridge formation in the HIV-1 MA structures (Fig. S15); and, SDS-PAGE showing the effect of His89 and E12 mutations on virus production (Fig. S16). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 20...

Targeted cancer immunotherapy with irradiated, granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study ( NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43–59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.