Contributions of Mamu-A*01 status and TRIM5 allele expression, but not CCL3L copy number variation, to the control of SIVmac251 replication in Indian-origin rhesus monkeys

Abstract

CCL3 is a ligand for the HIV-1 co-receptor CCR5. There have recently been conflicting reports in the literature concerning whether CCL3-like gene (CCL3L) copy number variation (CNV) is associated with resistance to HIV-1 acquisition and with both viral load and disease progression following infection with HIV-1. An association has also been reported between CCL3L CNV and clinical sequelae of the simian immunodeficiency virus (SIV) infection in vivo in rhesus monkeys. The present study was initiated to explore the possibility of an association of CCL3L CNV with the control of virus replication and AIDS progression in a carefully defined cohort of SIVmac251-infected, Indian-origin rhesus monkeys. Although we demonstrated extensive variation in copy number of CCL3L in this cohort of monkeys, CCL3L CNV was not significantly associated with either peak or set-point plasma SIV RNA levels in these monkeys when MHC class I allele Mamu-A*01 was included in the models or progression to AIDS in these monkeys. With 66 monkeys in the study, there was adequate power for these tests if the correlation of CCL3L and either peak or set-point plasma SIV RNA levels was 0.34 or 0.36, respectively. These findings call into question the premise that CCL3L CNV is important in HIV/SIV pathogenesis.

Figure 1. Intra-experiment reproducibility and inter-experiment variability in CCL3L copy-number determination.

(A) Intra-experiment reproducibility as determined using duplicates for each DNA sample from 84 rhesus monkeys. The results of two separate experiments are shown. (B) Inter-experiment variability. CCL3L copy numbers were determined by real-time qPCR using different amounts of input DNA. Correlations between the mean of the unrounded CCL3L copy number estimates from two separate experiments are shown. (C) Bubble plots showing the concordance between rounded CCL3L copy number estimates determined using different amounts of input DNA. The precision of each assay was determined by the rounded CCL3L copy number estimate of from reference sample, the A431 human cell line (black arrow).

Figure 2. Distribution of CCL3L copy number in Indian-origin rhesus monkeys.

Copy numbers of CCL3L genes were estimated using real-time PCR in 84 Indian-origin rhesus monkeys. (A) Frequency distribution of CCL3L copy number. Data are displayed three ways; combining all monkeys, dividing the monkeys into 2 cohorts: one Mamu-A*01− and the other Mamu-A*01+, or dividing the monkeys into 2 cohorts: one of animlas expressing only TRIM5 alleles 1–5 and the other of animals expressing at least 1 TRIM5 allele of the groups 6–11. The mean, variance, standard deviation (SD) and median of the copy number are shown. (B) Boxplots of CCL3L copy number in Mamu-A*01− and Mamu-A*01+ animals, and, in one of animlas expressing only TRIM5 alleles 1–5 and the other of animals expressing at least 1 TRIM5 allele of the groups 6–11. The comparisons were analyzed using the Mann-Whitney U test (two-tailed).

Figure 3. Distribution of CCL3L copy number in a cohort of Indian-rhesus monkeys.

Rhesus monkeys are divided into 4 groups according to their expression of Mamu-A*01 allele (A*01− and A*01+) and selected TRIM5 alleles (TRIM5 1–5: one expressing only TRIM5 alleles 1–5, TRIM5 6–11: the other expressing only TRIM5 alleles 6–11).

Figure 4. Lack of association of CCL3L CNV with virus replication in Indian-origin rhesus monkeys following SIVmac251 infection.

The plasma SIV RNA levels were assessed on days 14 and 70 following challenge, representing peak and set-point plasma SIV RNA levels, respectively. The monkeys were divided into 2 groups, one having CCL3L copy numbers below the median in this cohort of monkeys (black), and the other having CCL3L copy numbers above the median (red). These two groups of rhesus monkeys were subdivided into four separate groups according to their expression of Mamu-A*01 (− or +) and TRIM5 alleles (6–11 or 1–5). The association of CCL3L groups with plasma SIV RNA levels was assessed. The comparisons were analyzed using the Mann-Whitney U test (two-tailed).