Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation
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The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is necessary and sufficient to assemble non-infectious particles. Given that HIV-1 subverts many host proteins at all stages of its life cycle, it is essential to identify these interactions as potential targets for antiretroviral therapy.
This work demonstrates the use of proximity-dependent biotin identification (BioID) of host proteins and complexes that are proximal to the N-terminal domains of the HIV-1 Gag polyprotein. Two of the hits identified in the BioID screen were validated by immunoprecipation and confirmed the interaction of DDX17 and RPS6 with HIV-1 Gag.
Our results show that BioID is both a successful and complementary method to screen for nearby interacting proteins of HIV-1 Gag during the replicative cycle in different cell lines.
KeywordsProtein Synthesis Machinery Full Replication Cycle
Human immunodeficiency virus type 1
Proximity-dependent biotin identification
Acquired immunodeficiency syndrome
Interferon, IFI16, IFN-inducible protein 16
Astrocyte-elevated gene 1
DEAD-box RNA helicase 17
Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS), which specifically infects CD4+ T-cells, macrophages and dendritic cells. The integrated proviral DNA is transcribed to generate multiply and singly spliced and full-length (vRNA) messenger RNAs (mRNA). vRNA acts as both the mRNA for the Gag and Gag-Pol polyprotein precursors, and the genome that is encapsidated into assembling particles . The structural polyprotein Gag mediates virus assembly by trafficking to the plasma membrane, where it multimerizes to form spherical immature virus particles that are then released . During the budding process, the Gag precursor is cleaved by HIV-1 protease into mature Gag proteins p17 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC) and p6. The small size of the HIV-1 genome makes it reliant on host cellular machineries to replicate and interacts with host factors in order to neutralize host defenses and elicit pathogenesis.
Expression of BirA*-Gag
Identification of potential cellular interacting partners of BirA*-Gag
The host’s first line of defense against viral infection is the innate immune system whose activation leads to the production of proinflammatory cytokines and type I interferon (IFN) responses. The only protein classified within the GO immune system process was the IFN-inducible protein 16 (IFI16). Several classes of nucleic acid sensors have been implicated in HIV-1 RNA and DNA recognition  and IFI16 has been shown to colocalize and associated with lentiviral DNA in the cytoplasm of macrophages . Additionally, HIV-1 disease progression was affected by the genetic variation in IFI16 with a particular single-nucleotide polymorphism in its promoter region being associated with higher CD4+ T cell counts . A potential interaction between IFI16 and Gag may suggest a HIV-1 countermeasure to this important host defense.
Identified in Additional file 1: Table S1, Lyric (also known as Metadherin or astrocyte-elevated gene 1 (AEG-1)) was first identified as a HIV-1 inducible gene and has been suggested to play a role in a positive-feedback loop promoting HIV-1 replication  and has also been implicated in HIV-associated neuropathy [16, 15]. Engeland and colleagues recently demonstrated that Lyric interacts specifically with Gag and is incorporated into HIV-1 virions, where it is cleaved by the viral protease .
Validation of BirA*-Gag interactions
Recently, Ritchie and colleagues published a study in which they examined the interactions of Gag versus a MA-deleted Gag mutant in virus-producing HEK 293 T cells using BioID . Fifty cellular proteins were biotinylated by wild-type Gag, however, only one, Lyric overlapped with our list (Additional file 1: Table S1). The BioID approach is very useful for identifying weak and/or transient interactions (because biotinylation occurs before solubilization) and is amenable to temporal regulation. However, limitations include the neighbouring proteins having available primary amines to be biotinylated and consequently, the absence of biotinylation does rule out interaction or proximity . Our study taken together with the one performed by Ritchie and colleagues highlight the importance of the chosen location of the BirA* tag in the design of a construct, as their construct encoded BirA* within Gag between the MA and CA domains . The practical labeling radius of BirA* may be as small as 10 nm  and therefore insertion of BirA* adjacent to or within various Gag domains will affect the pool of biotinylated interacting proteins. Another limitation may be the choice of control pulldown, expressing BirA* alone may have inadvertently eliminated, in addition to non-specific interactors, some specific Gag interactors that also interact with BirA*. This short report thoroughly validated BioID as a rapid and reliable method of screening for both proximal and interacting proteins.
We would like to thank Drs. Greg Matlashewski and Alicia Bolt (McGill University) for invaluable technical expertise and Kyle Roux (Sanford Children's Health Research Center) for plasmids. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to AJM (MOP-38111, MOP-56974) and by The Canadian HIV Cure Enterprise Team Grant HIG-133050 (to A.J.M.) from the CIHR in partnership with the Canadian Foundation for HIV-1/AIDS Research and the International AIDS Society.
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