Evolution of replication efficiency following infection with a molecularly cloned feline immunodeficiency virus of low virulence

Abstract

The development of an effective vaccine against human immunodeficiency virus is considered to be the most practicable means of controlling the advancing global AIDS epidemic. Studies with the domestic cat have demonstrated that vaccinal immunity to infection can be induced against feline immunodeficiency virus (FIV); however, protection is largely restricted to laboratory strains of FIV and does not extend to primary strains of the virus. We compared the pathogenicity of two prototypic vaccine challenge strains of FIV derived from molecular clones; the laboratory strain PET(F14) and the primary strain GL8(414). PET(F14) established a low viral load and had no effect on CD4(+)- or CD8(+)-lymphocyte subsets. In contrast, GL8(414) established a high viral load and induced a significant reduction in the ratio of CD4(+) to CD8(+) lymphocytes by 15 weeks postinfection, suggesting that PET(F14) may be a low-virulence-challenge virus. However, during long-term monitoring of the PET(F14)-infected cats, we observed the emergence of variant viruses in two of three cats. Concomitant with the appearance of the variant viruses, designated 627(W135) and 628(W135,) we observed an expansion of CD8(+)-lymphocyte subpopulations expressing reduced CD8 beta-chain, a phenotype consistent with activation. The variant viruses both carried mutations that reduced the net charge of the V3 loop (K409Q and K409E), giving rise to a reduced ability of the Env proteins to both induce fusion and to establish productive infection in CXCR4-expressing cells. Further, following subsequent challenge of naïve cats with the mutant viruses, the viruses established higher viral loads and induced more marked alterations in CD8(+)-lymphocyte subpopulations than did the parent F14 strain of virus, suggesting that the E409K mutation in the PET(F14) strain contributes to the attenuation of the virus.

FIG. 1.

Quantification of proviral load and CD4/CD8 ratio in FIV-infected cats during the acute phase of infection. Three cats were inoculated intraperitoneally with either GL8₄₁₄ (○), PET_F14 (▿), or mock infected with PBS (□). (A) Proviral DNA load in PBMCs was estimated by real-time PCR (control cats were consistently negative and thus are not shown). Each point represents the mean proviral load of the three cats in the group (± standard error). (B) The CD4/CD8 ratio was estimated by flow cytometry. Results represent the mean CD4/CD8 ratio of the three study groups (± standard error).

FIG. 2.

Effect of FIV infection on CD4⁺ (A), CD8⁺ (B), and CD8α⁺β^low (C) lymphocyte subsets. The percentage of peripheral blood lymphoid cells expressing CD4 and CD8 was quantified in each of the three study groups, GL8₄₁₄ infected (○), PET_F14 infected (□), or mock infected with PBS (▿), by flow cytometry, and the absolute cell number of each subset was calculated from the results of hematological analysis. Results are expressed as the mean (± standard error) cell number (10⁹/liter); ∗ denotes a statistically significant difference (P = 0.046).

FIG. 3.

Quantification of proviral load in FIV-infected cats during the acute and chronic phases of infection. Three cats were inoculated intraperitoneally with either GL8₄₁₄ (open symbols) or PET_F14 (closed symbols), and the proviral DNA load in PBMCs was estimated by real-time PCR. The sequential proviral loads of each cat in the two groups are shown as % infected PBMCs.

FIG. 4.

Expansion of CD8α⁺β^low-lymphocyte subpopulations in FIV-infected cats. Contour plots of representative analyses of one cat from each group at 144 weeks postinfection (CON = control 612, GL8 = GL8₄₁₄-infected 612, and PET = PET_F14-infected 628). Boxed regions illustrate the analysis gates for each population.

FIG. 5.

Predicted amino acid sequence of the V3 -V6 region of the FIV envelope glycoprotein from revertant viruses (627_W135 and 628_W135) at 135 weeks postinfection. Lines under the sequence delineate the variable regions 3 to 6. Predicted sites for N-linked glycosylation (gray dots) and residues in the V3 loop affecting CXCR4 usage (↓) are marked. Sequences are shown relative to the parent molecular clone PET_F14 and to the GL8₄₁₄ molecular clone for comparison. Sequences were consistent between three independent clones.

FIG. 6.

Reduced fusogenicity of envelope glycoproteins from 627_W135 and 628_W135 viruses. env genes from PET_F14 (a), GL8₄₁₄ (b), 627_W135 (c), 628_W135 (d), and GL8_E407K (e) were subcloned into the vector VR1012 and were transfected into CXCR4-expressing AH927 cells. The empty vector VR1012 was transfected as a control (f). Forty-eight hours posttransfection the cells were fixed, stained, and examined by light microscopy. Panels a to f illustrate representative syncytia observed with each construct. The numbers of syncytia per field were enumerated; the data represent the mean number (n = 5) of syncytia per field ± standard error. CON, control.

FIG. 7.

Impaired growth of the 627_W135 and 628_W135 viruses in CXCR4-expressing feline cells. PET_F14, GL8₄₁₄, 627_W135, and 628_W135 viruses were used to infect AH927 cells that had been engineered to overexpress feline CXCR4. Infection was performed in the presence (open bars) or absence (hatched bars) of the CXCR4 antagonist AMD3100. Supernatants were collected at 9 days postinfection and were assayed for RT activity. Results represent the mean of duplicate samples and are expressed as picograms per milliliter of RT.

FIG. 8.

Quantification of proviral load (a), viral load (b), and CD8αβ^low (c) in FIV-infected cats during the acute phase of infection. Three cats were inoculated intraperitoneally with either PET_F14 (○), 627_W135 (▿), or 628_W135 (□). (a) Proviral DNA load in PBMC was estimated by real-time PCR. Each point represents the mean % infected PBMCs of the three cats in the group (+ standard error). (b) Viral load in plasma was estimated by real-time PCR. Each point represents the mean number of virions per milliliter of plasma from the three cats in the group (+ standard error). (c) Expansion of CD8α⁺β^low-lymphocyte subpopulations following infection with the three isolates, expressed as the ratio of CD8α⁺β^low cells to CD8α cells. Each point represents the mean ratio of CD8α⁺β^low to CD8α cells for the three cats in each group (+ standard error).