Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

Abstract

Background: Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.

Results: Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.

Conclusions: These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.

Figure 1

Virological and immunological characteristics of patient L. (A) Plasma viral load (diamonds) and CD4 + T-cell counts (triangles) of patient L from October 2005 till June 2007. An arrow indicates the probable time of HIV-1 superinfection. Biological clones were generated from PBMC samples collected in November 2005 and January 2006, respectively. (B) NJ tree constructed with representative nucleotide sequences derived from HIV-1 env-V3 obtained from plasma collected from patient L. Separate clusters formed by strains B1 and B2 are indicated. Env sequences from biological clones are indicated with clone numbers. Symbols in the tree correspond to samples from November 2005 (circles), January 2006 (diamonds) and April 2006 (squares). Reference sequences were HIV-1 strain HXB2 and subtypes C and D strains, respectively. The scale bar indicates the nucleotide distance between the sequences (as calculated with the Tamura-Nei method [59]).

Figure 2

Virological and immunological characteristics of patient P. (A) Plasma viral load (diamonds) and CD4 + T-cell counts (triangles) of patient P from March till December 2006. An arrow indicates the probable time of HIV-1 superinfection. A second arrow indicates the start of highly active antiretroviral therapy (HAART) in November 2006. Biological clones were generated from PBMC samples collected in March 2006 (31 st of March) and August 2006, respectively. (B) NJ tree of HIV-1 env-V3 nucleotide fragments obtained from plasma collected from patient P. Separate clusters comprised of strains B3 and B4 are indicated. Env sequences from biological clones are indicated with clone numbers. Symbols in the tree correspond to samples from March 2006 (circles), August 2006 (diamonds) and November 2006 (squares). Reference sequences were from HIV-1 strain HXB2, and subtypes C and D strains, respectively. The scale bar indicates the nucleotide distance between the sequences (as calculated with the Tamura-Nei method [59]).

Figure 3

PBMC gene expression patterns of HIV-1 biological clones. mRNA expression levels in PBMC's infected with the HIV-1 biological clones B1.1, B1.3, B2.5 (patient L) and B3.1, B4.2, and B4.4 (patient P), as well as uninfected PBMC's were analysed with the RT²Profiler™ PCR Array Human Inflammatory Cytokines and Receptors (SABiosciences). Cultures were infected with HIV-1 at an MOI of 0.05. After two hours, the inoculum was removed by centrifugation. Total RNA was isolated 6 hours after infection. Experiments were performed in triplicate. Expression profiles were analysed with the GCNPro™ (Gene Network Central) software [68]. Clustering of the gene expression profiles induced by the HIV-1 clones is shown for a selection of genes from a representative experiment. Green colour indicates increased mRNA expression, red colour indicates decreased mRNA expression compared to the uninfected PBMC's.

Figure 4

HIV-1 LTR and Tat sequences. (A) Nucleotide sequences of the LTR region from clones B1.1, B1.3, B2.3 (primary and superinfecting strain from patient L, respectively), and clones B3.1, B4.2, and B4.4 (primary and superinfecting strain from patient P, respectively). Sequences were aligned using the HXB2 sequence (GenBank acc. no. K03455) as reference. Binding sites for transcription factors and the two insertions found in clone B1.1 (type I and type II) have been boxed. A NF-κB/NFAT binding site immediately followed by an YY1 binding site found only in clone B1.1, are indicated. The TATA-box and the TAR region (nt 504-555) have been underlined. A destabilizing T→C mutation in the TAR hairpin region in clone B2.3 is boxed. The polyA hairpin (nt 556-602) is shown in bold, a box indicates the destabilizing TT→CA mutation in clone B3.1. (B) Translated amino acid sequences are shown for HIV-1 Tat. Sequences have been aligned with the HXB2 sequence. Clone numbers are indicated. Strains B1 and B2 are the first and superinfecting virus from patient L, respectively. Strains B3 and B4 are the first and superinfecting virus from patient P, respectively. The Tat T23 N and F32 L mutations in strain B1 associated with increased and decreased Tat activity have been boxed.

Figure 5

Structure of the LTR polyA hairpin. Predicted structure of the LTR polyA hairpin region of the HIV-1 reference strain HXB2 and clones B3.1, B4.2 and B4.4. The free energies of the stem-loop structures were calculated with the Zuker algorithm as available at the mfold webserver for nucleic acid folding and hybridization prediction [69], the ΔG values are presented in kilocalories per mole. A box indicates the UU→CA change in clone B3.1.

Figure 6

HIV-1 Gag p1-p6 and Env-V3 sequences. Translated amino acid sequences are shown for HIV-1 Gag p1-p6 region (panel A), and env-V3 (panel B). Sequences were aligned with the HXB2 reference sequence. Clone numbers are indicated. Strains B1 and B2 are the first and superinfecting virus from patient L, respectively. Strains B3 and B4 are the first and superinfecting virus from patient P, respectively. The 11 aa PTAPP repeat in clone B3.1 in Gag-p6 has been boxed. The 11^th and 25^th amino acid residues in Env-V3, associated with CXCR4 coreceptor use when positively charged, are indicated.

Figure 7

Transcriptional activity of the LTR promoter sequences. Transcriptional activity of the HIV-1 LTR promoter sequences from HIV strains B(LAI), B1, B2, B3, and B4, compared with the empty vector (pGL3-basic) in a dual firefly/renilla luciferase assay. LTR fragments cloned from clones B1.1/B1.3 and B4.2/B4.4 are identical in sequence, so only strain names are indicated. Transcriptional activity of the luciferase gene was tested in the presence of increasing concentrations of Tat. The value is the average of three independent measurements; standard deviations are indicated.