Glycosylation is a fundamentally important process in HIV and SIV biology. It is one of the major mediators of virus binding [1
], is required for proper processing of the viral envelope proteins [3
] and is key in immunologic escape [4
]’. From a vaccination standpoint, differences in glycosylation can change how antigen presenting cells interact with the vaccine [45
] , and obviously change the nature of the immune responses.
The SIV strains chosen for this study have been widely utilized in the most relevant animal models for HIV pathogenesis and vaccine research [20
]. Therefore an understanding of the fundamental differences between these viruses may help to explain some of the differences in disease progression observed between the non-human primate models of HIV disease[46
]. Regarding proteins differentially expressed in these preparations, although we cannot attribute the presence of absence of these proteins to virions or microvesicles, it is important to note that these proteins themselves can elicit immune responses. For example it has been shown that animals immunized with HLA-DR can confer resistance against challenge with SIV[52
]. Therefore even the basic information obtained from a 2DE-DIGE experiment () may be revealing.
Although our success in identifying glycosylation site usage is limited in comparison to more recent studies using recombinant sources of ENV protein [9
], we have shown glycosylation site usage in regions of the virus used in immune evasion and CD4 binding (V3) in physiologically relevant sample sources and did obtain high sequence coverage for gp120 (Supplemental Tables 1
). We were not surprised at the limited observation of gp120 glycosylation sites from our 1D gel experiments since we were only able to load 25ug per lane or 10 fold less gp120 than on a 2D gel. Direct assessment of spots from a 2D gel using MALDI-TOF/TOF or FT-ICR instrument and direct comparison of fully glycosylated and de-glycosylated spectra and the use of 018
labeled water to minimize the likelihood of false positives would likely increase the identification of putative glycosylation sites as recently reported [53
]. The low extraction efficiency associated with gel-based methods is also likely responsible for the low number of modified peptides observed. It is therefore likely that if HPLC methods were used to first purify gp120 from the virus preparations [36
] then much higher sequence coverage could be obtained; however, information on post-translational modifications imparted by different oligosaccharides would be lost, as these are best visualized by a 2D gel method. Further this method is extremely rapid as results may be interpreted directly at the 2D gel level.
Although many studies have used site directed mutagenesis to change potential glycosylation sites of HIV and SIV [54
] , there is a paucity of findings of actual in vivo
glycosylation sites due to the very large amounts of virus required to make this determination. Our experimental pipeline allows the determination of the actual usage of glycosylation sites in complex mixtures of virions. Furthermore, we can also address the issues of relative ENV incorporation and ENV heterogeneity in a sample. For instance, it has been estimated that HIV virions contain 7-10 trimers of gp120 based upon the amount of ENV protein [36
]. However, is this ENV homogenous or heterogeneous with regard to glycosylation? The data presented in this study show that the heterogeneity observed with regard to isoelectric point is likely due to compositional changes in the N-linked carbohydrates rather than site utilization given the relative uniformity of molecular masses observed. Further studies will be required to determine the exact oligosaccharide composition for this viruses used in this study.
Finally, the role of O-GlcNAcylation in cell biology has only begun to be recognized [55
]. Our results show that HLA-DR is O-GlcNAc modified (). Many reports in the literature describe the specific incorporation of HLA-DR into HIV [38
], but the specifics of this mechanism are still unknown. Although the role of the invariant chain in HLA-DR shuttling to endosomes has been well described [56
], it is unclear what properties O-GlcNAc modifications of the cytoplasmic tail (O-GlcNAc transferase is localized to the nucleus and cytoplasm) of HLA-DR might confer. The immunological implications of our findings may potentially shed light on the specific nature of HLA incorporation into viral particles. These findings again raise the question of the significance of host protein incorporation into virion particles [57
]. Using this methodology, one can track the presence and absence of different host proteins at a global level, and determine if there are differentially glycosylated forms of host proteins that are more highly enriched than in the cell, and potentially have different functions than their unmodified counterparts. The pilot results presented here suggest there will be dramatic differences in host protein composition depending on what cell type the virus is isolated from and the status of the cell at that time ().
Collectively, these data will hopefully help to overcome some specific challenges facing vaccine design regarding antigen quality control, and help to provide an alternative method of validation for studies examining viral sequences and correlation of viral sequence with pathogenicity [58
]. This is important since examination of the nucleic acid sequence information of virions alone cannot consider differences present at the level of glycosylation (or other post translational modifications observable by 2DE) between different infected target cells (). Since glycosylation status can change based upon the state of the host cell [59
], be induced by inflammation [60
], or be unique to different cell types [33
], such studies would overlook differences at the level of this important post-translational modification. Due to this limitation, even very well controlled studies that conclude that the ENV glycoprotein is not responsible for changes in pathogenicity may have overlooked the possibility that differences in glycosylation in ENV may be responsible for the effect [29
]. By applying our methods retrospectively to viruses that have been extensively studied and used in vaccine studies, like the SIV strains used in this study [21
], we may be able to better understand the types of results that were obtained, and potentially understand why certain vaccine trials failed to protect.