Biological determinants of HIV infection : studies of viral evolution during disease progression in children and adults

Abstract

Coreceptor usage of primary HIV-1 isolates was analysed in relation to their biological phenotype and the severity of HIV-1 infection in the patient. The indicator cell lines, U87 glioma cells engineered to express CD4 and one of the chemokine receptors CCR1, CCR2b, CCR3, CCR5 or CXCR4, were infected with a panel of well-characterized primary HIV-1 isolates. Receptor usage was determined by monitoring cell cultures for cytopathic effects and p24 antigen production. Strikingly, all slow/low viruses used the CCR5 receptor (R5), whereas all rapid/high viruses used CXCR4, and in most cases also CCR5, in some cases also CCR2b or CCR3. Only a few isolates used solely CXCR4 (X4). In all cases, tropism for MT-2 cells, a previously used indicator cell line, correlated with CXCR4 usage. This distinct pattern of coreceptor usage was closely associated with the biological phenotype and not the genetic subtype (A, B and D) of the virus. However, the biological phenotype of subtype C isolates obtained from Ethiopian late-stage AIDS patients, showed a few unexpected features. Isolates replicated slowly in peripheral blood mononuclear cell cultures and did not infect established cell lines. All isolates used CCR5 as coreceptor, yet no productive infection could be established in primary macrophages. On the basis of these data, we suggest that additional factors than viral phenotype may govern the pathogenic potential of subtype C isolates.

Coreceptor usage of eleven primary HIV-2 isolates obtained from West African individuals with varying severity of HIV-2 infection was also tested. As for HIV-1, all but one HIV-2 isolate used CCR5 and two isolates from patients with late-stage disease used CXCR4. However, the CCR5-using HIV-2 isolates were promiscuous in their receptor usage and used at least one additional receptor (CCR1, CCR2b, CCR3 or BOB/ GPR15) indicating that multiple coreceptor usage and HIV-2 virulence are not correlated. Evolution of coreceptor usage during disease progression was analysed in sequential HIV-1 isolates obtained from nine children. In all cases, a virus with R5 phenotype was present in the early phase of infection. The change in phenotype over time was strictly associated with the acquisition of the ability to use CXCR4. Clinical and immunological decline were coherent with the shift of phenotype, appearance of CXCR4-using virus and resistance to inhibition by beta-chemokines. This indicates that escape from the inhibitory effect of beta-chemokines is closely related with the phenotype switch.

It has been suggested that R5 viruses are preferentially transmitted and that macrophages are particularly important vehicles of HIV-1 in sexual transmission. We therefore wanted to see if also X4 viruses could infect macrophages. We found that R5 and X4 (including the T-cell line adapted isolate IIIB), as well as dual tropic R5X4 or R3X4 viruses could establish a productive infection in monocyte-derived macrophages. This shows that macrophage tropism is a general property of HIV-1 isolates. We investigated the genetic polymorphism of sequential primary isolates obtained from slow and fast progressing children by using the DNA heteroduplex mobility assay. In general, the first and second variable (V 1 /V2) and second constant (C2) domains of the gp120 envelope region, but not V3, showed heterogeneity early after birth. Slow progression was associated with a greater degree of genetic polymorphism. DNA sequencing revealed that X4 viruses have an increased net charge in the V2C2 and V3 region compared to CCR5-using (mono- and dualtropic) viruses. Our findings call for further studies of the whole envelope region to determine residues critical for coreceptor binding.