Viral sequence diversity is the Achilles' heel of traditional vaccine approaches, as exemplified by influenza vaccinology. The influenza vaccine must change yearly in anticipation of the predominant circulating viral strains, which differ from one another by only 1 to 2% (1
). In contrast, circulating human immunodeficiency virus type 1 (HIV-1) strains differ from each other by 20% in the more conserved proteins and up to 35% in the Env protein (1
). This enormous sequence diversity is a major stumbling block to the development of a conventional HIV-1 vaccine. Indeed, a neutralizing antibody-based vaccine has proven elusive, in part due to the extremely high sequence variation of the HIV-1 Env protein (3
). While CD8+
T cell-based vaccines directed against more-conserved viral proteins have shown some ability to blunt viral replication (4
), they are also hindered by HIV-1 sequence variation (6
). Even minor variations in sequence can have a dramatic effect on CD8+
T cell efficacy, as just one amino acid change can abrogate recognition and suppression of virus replication (7
). Additionally, HIV-1 rapidly mutates to escape effective cytotoxic T lymphocyte (CTL) responses, thereby raising the possibility that any effective vaccine will only have a transient effect (6
). On a population level, CD8+
T cells are driving HIV-1 evolution toward fixation of epitope escape variants that are more likely to evade the immune responses of the population in which they circulate (12
). Therefore, a successful HIV-1 vaccine must overcome the formidable challenge of HIV-1 sequence diversity. In this study, we sought to determine whether invariant self-antigens overexpressed within an HIV-1-infected cell could act as potential immune targets for vaccine development. Specifically, we examined HIV-infected patients and simian immunodeficiency virus (SIV)-infected macaques for the presence of APOBEC-specific T cell responses.
Mammalian host cells have developed intrinsic mechanisms to prevent lentiviral replication, as well as to maintain genomic stability by restricting the movement of retroelements. These mechanisms include postentry interference by tripartite motif-containing protein 5α (TRIM5α), transcriptional silencing through DNA methylation, posttranscriptional silencing via RNA interference, tetherin, and mutational inactivation of elements in the course of their retrotransposition cycle by cellular cytosine deaminases (14
). The apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases consists of 7 members (APOBEC3A to APOBEC3H) in humans, all of which are encoded on the same gene cluster of chromosome 22. These enzymes are expressed in the majority of human cells and have been extensively studied since the discovery that APOBEC3G acts as a viral restriction factor in HIV-1 infection (16
). Since that time, multiple additional APOBEC3s have been implicated in HIV restriction (17
APOBEC3G is packaged into budding HIV-1 virions through an interaction with the nucleocapsid region of HIV-1 Gag, and its proclivity for binding single-stranded nucleic acids facilitates this process (20
). Following HIV-1 infection of a target cell, the viral RNA genome is uncoated, and reverse transcriptase generates a single strand of DNA complementary to the viral genome. During this transcription process, reverse transcriptase also degrades the viral RNA strand through its RNase H activity, leaving only the single strand of cDNA (20
). APOBEC3G exerts its enzymatic activity on this single-stranded DNA, mutating cytosines to uracils. These extensive mutations can cause degradation of the viral genome or inhibit viral replication due to altered reading frames.
Alternatively, APOBEC3G has been shown to inhibit HIV-1 replication independent of its enzymatic function. First, it can impair reverse transcriptase activity by blocking the binding of tRNALys3
(the primer that initiates reverse transcription). Second, it can stifle the cleavage of tRNALys3
from the single-stranded DNA intermediate, leading to aberrant viral DNA ends (19
). Finally, it can bind to HIV-1 integrase, a component of the viral preintegration complex, suggesting that it may obstruct nuclear homing of provirus (19
HIV-1 expresses 6 accessory proteins that are integral to its replication and persistence. One of these accessory proteins is the viral infectivity factor (Vif) protein. The function of Vif was initially elucidated from experiments showing that Vif-deficient HIV could replicate in certain cells (permissive), but not in others (nonpermissive). Infection of a hybridoma of these two cell types with Vif-deficient HIV-1 yielded noninfectious virions, implying that a host cellular restriction factor was obstructing further infections.
Subsequent analysis of two highly related cell lines, one permissive and the other nonpermissive, using a cDNA subtraction strategy revealed that this cellular restriction factor was APOBEC3G (22
). Since this discovery, Vif has been shown to ubiquitinate APOBEC3G, leading to degradation by the proteasome. Indeed, the levels of APOBEC3G are drastically reduced in virion-producing cells infected with HIV-1, and APOBEC3G is not packaged into progeny virions in the presence of Vif (23
). Vif has been shown to inhibit APOBEC3G and APOBEC3F, but not APOBEC3B (19
). Thus, although the physiological role of APOBEC3 proteins is not fully understood, they represent an ancient form of antiviral or antiretroelement defense in mammals (19
The proteasome is an important first step in the antigen presentation pathway for HLA class I peptide epitopes recognized by CD8+ T cells. We hypothesized that HIV-1 infection would give rise to CD8+ T cell responses against APOBEC3 proteins, due to increased Vif-triggered proteasomal processing and HLA class I presentation. In a cell not infected by HIV-1, there would be limited presentation of APOBEC3-derived peptide epitopes on the surface as part of the normal repertoire of self-antigens presented by major histocompatibility complex class I (MHC-I) molecules.
The immense sequence diversity of HIV-1 remains a major roadblock to a prophylactic HIV-1 vaccine. Targeting CD8+ T cells to HIV-1-infected cells by their higher expression of APOBEC3 antigens may be a novel way to circumvent the obstacle presented by viral sequence diversity. Here, we present the first data showing APOBEC3-specific T cell responses in both HIV-1-infected humans and SIV-infected rhesus macaques.