Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.The envelope glycoprotein complex of the human immunodeficiency virus type 1 (HIV-1) is involved principally in virion attachment to target cell surfaces and in the entry process (15, 18, 27, 29, 52). Envelope glycoproteins (Env) are initially translated as a gp160 precursor glycoprotein, which is then processed during its trafficking through the secretory pathway, to yield a surface subunit gp120 noncovalently attached to a transmembrane subunit gp41. During HIV-1 assembly, Env proteins are incorporated at the surface of the viral particle as a trimeric structure consisting of three gp120/gp41 dimers (59, 62).The gp41 consists of an ectodomain, a hydrophobic transmembrane anchor, and a cytoplasmic tail (CT). Lentiviruses, including HIV-1 and simian immunodeficiency virus (SIV), are unusual in having a transmembrane subunit with much longer CTs (∼150 amino acids) than most other retroviruses (20 to 50 amino acids) (27). Early studies with T-cell laboratory-adapted HIV-1 mutants showed that the gp41 CT region played an important role in regulating Env functions, the incorporation of Env into virus particles and, consequently, viral replication (16, 21, 35, 63). The integrity of the gp41 CT thus appears to be crucial for replication in primary T cells, macrophages, and in many transformed T-cell lines (1, 44). Viral variants with truncated gp41 are rarely isolated from infected patients. One study reported the isolation of a CD4-independent variant harboring a sharply truncated CT (64). However, this atypical isolate existed as a minority variant in the original quasispecies of the patient (54). SIV variants with truncated CTs obtained in cell culture in vitro have also been shown to revert rapidly (to full-length CT) when introduced into macaques (39). These observations indicate that the long CTs of lentiviruses, such as HIV-1 and SIV, have functions specific to viral replication and persistence in vivo.Two groups of conserved sequence motifs have been identified in the gp41 CT that are likely to be involved in its functions. The first group, involved in regulating the intracellular trafficking of Env, includes a membrane-proximal tyrosine-based endocytic motif, Y₇₁₂SPL, (9, 47); a diaromatic motif, Y₈₀₂W₈₀₃, implicated in the retrograde transport of Env to the trans-Golgi network (8), and a C-terminal dileucine motif recently identified as a second endocytic motif (7, 10, 60). We have also provided evidence for the existence of additional as-yet-unidentified signals in studies of primary HIV-1 (34). The second group of motifs consists of three structurally conserved amphipathic α-helical domains: lentivirus lytic peptides 1, 2, and 3 (LLP-1, LLP-2, and LLP-3) (11, 17, 33). LLP domains have been implicated in various functions, including Env fusogenicity and the incorporation of Env into HIV-1 particles (28, 32, 43, 45, 50, 61).Several lines of evidence suggest that Env incorporation requires direct or indirect interactions between the matrix domain of the structural protein precursor Pr55^Gag (matrix) and the gp41 CT during HIV-1 assembly. This possibility was first suggested by the observation that HIV-1 Env drives the basolateral budding of Gag in polarized cells (37, 48). A direct interaction between the matrix and a glutathione S-transferase fusion protein containing Env CT was subsequently observed in vitro (13). Synthetic peptides corresponding to various domains of the gp41 CT have also been shown to interact directly with Pr55^Gag molecules (26). Furthermore, effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by studies based on T-cell laboratory-adapted virus mutants (19, 40, 43). Finally, the cellular protein TIP47 was recently implicated in Env incorporation, based on its ability to bind both the matrix protein and the gp41 CT (38).In a previous study describing the evolutionary dynamics of the glycan shield of HIV-1 Env, we identified a patient (patient 153) for whom the 15 env clones obtained during primary infection (early stage) encoded full-length Env, whereas the 15 env sequences from the HIV-1 present 6 years later (late stage) encoded truncated gp41 CTs (14). These late-stage sequences contained a deletion introducing an in-frame stop codon, resulting in a 20-amino-acid truncation of the Env. Note that, unlike a point mutation, this deletion cannot easily revert to the full-length form. Such a deletion affecting various known motifs of the gp41 CT would be expected to impair viral replication. However, the plasma viral load measured in patient 153 demonstrated that the virus had retained its ability to replicate.In the present study, we explored the molecular mechanisms by which a primary HIV-1 maintained its capacity to replicate efficiently in this patient and demonstrated for the first time the occurrence of matrix and Env coevolution in vivo, providing insight into the ability of HIV-1 to overcome major structural alterations.