Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants

The entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by envelope glycoproteins on the surface of the viral particle. Although most strains of HIV-1 are able to infect primary lymphocytes, most molecularly cloned viruses are capable of infecting either primary macrophages or T-lymphoid cell lines, but not both cell types (64, 67). T-cell line-tropic strains are able to induce syncytia in primary lymphocytes and are designated syncytium-inducing strains, whereas most macrophage-tropic isolates display the non-syncytium-inducing phenotype. Additionally, isolates able to infect T-cell lines replicate more rapidly and to higher levels in primary lymphocytes (‘‘rapid, high’’ phenotype) than macrophage-tropic isolates, which replicate more slowly and to lower levels (‘‘slow, low’’ phenotype) (65). The emergence of T-cell line-tropic syncytium-inducing isolates in infected patients correlates with the progression of HIV-1 infection as characterized by declining CD4 lymphocyte counts and the appearance of clinical symptoms of AIDS (63, 72, 73), whereas macrophage-tropic isolates that display the non-syncytium-inducing phenotype are generally found within the first few months after infection and persist throughout the course of infection (4, 22, 35, 43, 58, 63). It appears that the uncompromised immune system suppresses high-replicating SI variants present in the inoculum, but slow-replicating non-syncytium-inducing variants avoid eliciting an effective immune response by their ability to infect macrophages. However, it is unclear whether the emergence of T-cell line-tropic isolates is a result of host immune system deterioration or whether these isolates are the causative agent of immune dysfunction. Many studies have examined the viral determinants necessary for infection of primary macrophages (13, 30, 54, 66, 67, 78–80). By analysis of full-length chimeric proviral clones between strains of HIV-1 either capable or incapable of infecting macrophages, a region of the Env protein containing the third hypervariable region (V3) has been identified as a minimal macrophage-tropic determinant (30), although additional viral sequences within the Env protein (67, 80) or accessory proteins Vpr and Vpu (79) are required for maximum infection. The V3 loop is a well-characterized domain of 34 to 37 amino acids that is formed by a disulfide bond between cysteine residues 296 and 331 (38). This region of the Env protein serves as a primary target for neutralizing antibodies, functions as a fusion domain, and regulates the sensitivity of HIV-1 to soluble CD4 (sCD4) (17, 19, 25, 30–32, 52, 59, 77, 83), although it is distinct from the domain of the envelope protein important for the initial interaction with the cellular receptor CD4 (55). The V3 loop plays an important role in regulating HIV-1 infection of other cell types in addition to macrophages, such as T-lymphoid cell lines. However, the V3 loop by itself is insufficient for conferring infection of T-cell lines. Nevertheless, substitution of a T-cell line-tropic V3 loop with a macrophage-tropic V3 loop results in loss of infection of T-cell lines (9, 30, 67). This implies that interaction of the V3 loop with other portions of the Env protein is required to establish the proper conformation necessary for infection. Previous work has suggested a functional interaction between the variable region (V3) and the fourth conserved domain (C4) lying between the fourth and fifth hypervariable regions (2, 49, 50, 86). The C4 domain constitutes part of the CD4-binding site and has also been implicated as important for association with the gp41 transmembrane glycoprotein (16, 27, 33, 34, 37, 55). In the present study, we have identified two amino acid residues lying within the C4 domain that are required in combination with the V3 loop to establish infection in T-cell lines. Identification of a minimal T-lymphoid cell line infection determinant. In order to examine the molecular determinants necessary for T-lymphoid cell line tropism, chimeric proviruses were constructed between two HIV-1 strains differing in their ability to infect T-cell lines. Both strains are capable of productively infecting peripheral blood lymphocytes (PBLs) (Fig. 1A). The following procedure was used. Chimeric HIV-1 clones were constructed from the T-cell line-tropic clone NLHX and the macrophage-tropic clone YU-2 (39). NLHX was derived from the NL4-3 clone by subcloning of the 2.7-kb SalI-BamHI fragment (nucleotides 5785 to 8474) from HXB2 into the full-length NL4-3 clone, which corrects a vpr defect and results in an intact vpr open reading frame (1). This construct is designated as HX. A 3.2-kb EcoRI-XhoI fragment (nucleotides 5742 to 8896) from the macrophage-tropic YU-2 proviral clone was inserted into NL4-3 and is designated as YU. Construct YH-GG was generated by exchanging the 0.6-kb BglII-BglII fragment (nucleotides 7040 to 7620) from HXB2 into construct YU, and constructs YH-GP and YH-PG were generated by reciprocal exchanges of 1.1-kb PpuMI-PpuMI fragments (nucleotides 7323 to 8396) between constructs YU * Corresponding author. Mailing address: Washington University School of Medicine, Box 8069, 660 S. Euclid, St. Louis, MO 63110. Phone: (314) 362-8836. Fax: (314) 454-1792. † Present address: Abbott Laboratories, Abbott Park, IL 60064-3500.