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. 1998 May;72(5):4478-84.
doi: 10.1128/JVI.72.5.4478-4484.1998.

Determinants of entry cofactor utilization and tropism in a dualtropic human immunodeficiency virus type 1 primary isolate

R J Smyth  1 , Y Yi, A Singh, R G Collman
Affiliations

Determinants of entry cofactor utilization and tropism in a dualtropic human immunodeficiency virus type 1 primary isolate

R J Smyth et al. J Virol. 1998 May.

Abstract

Human immunodeficiency virus type 1 strain 89.6 is a dualtropic isolate that replicates in macrophages and transformed T cells, and its envelope mediates CD4-dependent fusion and entry with CCR5, CXCR-4, and CCR3. To map determinants of cofactor utilization by 89.6 and determine the relationship between cofactor use and tropism, we analyzed recombinants generated between 89.6 and T-cell-tropic (HXB) or macrophage-tropic (JRFL) strains. These chimeras showed that regions of 89.6 env outside V3 through V5 determine CXCR-4 utilization and T-cell line tropism as well as CCR5 utilization and macrophage tropism. However, the 89.6 env V3 domain also conferred on HXB the ability to use CCR5 for fusion and entry but not the ability to establish productive macrophage infection. CCR3 use was conferred on HXB by 89.6 env V3 or V3 through V5 sequences. While replacement of the 89.6 V3 through V5 region with HXB sequences abrogated CCR3 utilization, replacement of V3 or V4 through V5 separately did not. Thus, CCR3 use is determined by sequences within V3 through V5 and most likely can be conferred by either the V3 or the V4 through V5 domains. These results indicate that cofactor utilization and tropism in this dualtropic isolate are determined by complex interactions among multiple env segments, that distinct regions of the Env glycoprotein may be important for utilization of different chemokine receptors, and that determinants in addition to cofactor usage participate in postentry stages in the virus replication cycle that contribute to target cell tropism.

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Figures

FIG. 1
FIG. 1
Parental and chimeric viruses utilized in this study. (A) Recombinants were generated in the 3′ hemigenome subclone by using BglII and MstII restriction sites in the env gene that flank the V3 and V4 through V5 domains. Chimeric 3′ subclones were cotransfected with the corresponding 5′ subclones to generate the recombinant viruses. (B) Nomenclature of the viruses, indicating the backbone, env region exchanged, and source of inserted sequences. Tropism was defined on the basis of productive replication in primary macrophages, transformed MT-2 cells, or both, and strains were segregated into those that typically produced p24 antigen levels of ≥10 ng/ml in MDM or MT-2 cells and those that produced ≤0.5 ng of p24 antigen/ml, as determined by enzyme-linked immunosorbent assay (Dupont, Wilmington, Del.). All viruses replicated in primary PBL. Infections and cell culture conditions have been described previously (23).
FIG. 2
FIG. 2
HIV-1 entry mediated by CCR5 and CXCR-4 in transfected quail cells. CD4-expressing QT6.CD4 quail fibrosarcoma cells were transfected by calcium phosphate with CCR5, CXCR-4, or control plasmids and then infected the following day with DNase-treated cell-free virus stock by using 25 ng of p24 antigen of each virus. Seventy-two hours later, the cells were washed and lysed in DNA lysis buffer. One-tenth of the lysate was amplified by PCR using primers that detect a 430-bp U3 through U5 region of the HIV-1 LTR, followed by Southern blotting using an internal oligonucleotide probe as described previously (16, 34). QT6.CD4 cells transfected with vector only served as a negative control.
FIG. 3
FIG. 3
Cell-cell fusion mediated by chimeric Env glycoproteins. Fusion between Env-expressing cells and CD4- and cofactor-expressing cells was determined by a content mixing assay using the luciferase reporter gene as previously described (16, 34). Chimeric env genes were subcloned into plasmids downstream of the T7 promoter. These or parental env genes (driven by T7 [HXB] or vaccinia early/late promoter [89.6]) were then transfected into 293 cells that had previously been infected with recombinant vaccinia virus vTF1.1, which expresses T7 polymerase. The Env-expressing 293 cells were then mixed with QT6.CD4 cells that were cotransfected with a T7-luciferase plasmid and plasmids expressing CCR5, CXCR-4, or control vector. Cell-cell fusion was determined by luciferase activity in cell lysates 16 h later and is expressed as a percentage of that seen with CCR5 (for 89.6-based Envs) or CXCR-4 (for HXB-based Envs). Data represent means ± standard errors of the mean from two to four wells per strain in each of three separate experiments.
FIG. 4
FIG. 4
Viral entry into primary macrophages and transformed MT-2 cells. (A) Seven-day-old MDM were purified and maintained as described previously (23) and then infected with DNase-treated virus stocks by using 25 ng of p24 antigen per virus. Two days later, cells were lysed and subjected to PCR amplification using primers directed against the HIV-1 LTR followed by Southern blotting (top), or using primers directed against gag sequences followed by staining with ethidium bromide (bottom). The LTR primers have been described previously (16), and gag primers (5′-GGTACATCAGGCCATATCACC-3′ and 5′-TGACATGCTGTCATCATTTCTTC-3′) amplify a 627-bp region of gag DNA sequence. (B) MT-2 cells were infected with DNase-treated virus stocks as described above. Two days later, they were lysed and subjected to PCR amplification using primers directed against the HIV-1 LTR, followed by Southern blotting.
FIG. 5
FIG. 5
HIV-1 entry mediated by CCR3 in QT6 cells. Quail QT6.CD4 cells were transfected with a plasmid encoding CCR3 or vector alone and then infected with DNase-treated virus stocks by using 25 ng of p24 antigen per virus. Forty-eight hours later, they were lysed and subjected to PCR amplification in order to detect viral reverse-transcription products by using primers directed against the HIV-1 LTR, followed by Southern blotting. QT6.CD4 cells transfected with vector only served as a negative control.
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