Download Page 1 Supplementary information Lifespan of effector memory CD4

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Supplementary information
Lifespan of effector memory CD4+ T cells determined by replicationincompetent integrated HIV-1 provirus
Hiromi Imamichi, Ven Natarajan, Joseph W. Adelsberger, Catherine A. Rehm,
Richard A. Lempicki, Biswajit Das, Allison Hazen, Tomozumi Imamichi, and
H. Clifford Lane
Supplemental Figure 1.
The protease sequences containing the W42Stop provirus collected from 19932000 (98 sequences) from effector memory CD4+ T cells of subject 1
Supplemental Figure 2.
Gating strategy used for FACS-sorting of naïve, central memory and effector
memory CD4+ T cells from subject 1
Supplemental Figure 3.
Exon-Intron structure of the human SMC5 gene and the locations of PCR primers
used in the present study
Supplemental Table 1
Primer pairs used for the inverse PCR and the confirmatory PCR
Supplemental information for Methods
Site-directed mutagenesis and preparation of virus stocks
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Supplemental Figure 1. The protease sequences containing the W42Stop
provirus collected from 1993-2000 (98 sequences) from effector memory
CD4+ T cells of the study subject were identical without a single nucleotide
substitution. HIV-1 HXB2 is used as a reference. The nucleotide sequence is
displayed at the top; and the corresponding amino acid sequence is shown below.
Darkened letters in red in the sample sequences indicate changes from the
reference sequence. The position of TGG (W)-to-TAG (Stop) change is
highlighted in yellow. An asterisk indicates a pre-termination stop codon.
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Supplemental Figure 2. Gating strategy used for FACS-sorting of naïve,
central memory and effector memory CD4+ T cells from subject 1. Doublet
discrimination gating was used prior to subsequent gating on CD3, CD4,
CD45RO and CD27. Two sets of plots representing the sorting experiments for
cryopreserved cells from 9 different time points are shown. The purity of the cells
is indicated in the plots.
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Supplemental Figure 3. Exon-Intron structure of the human SMC5 gene and
the locations of PCR primers used in the present study. The human SMC5
gene is located on chromosome 9q12.21 and composed of 25 exons. The HIV-1
provirus is integrated in the opposite orientation to the SMC5 gene. The
integration site is located within an intron between exons 11-12 of the SMC5.
Primers are numbered according to the HIV-1 HXB2 sequence.
Supplemental Table 1
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(1) Primer pairs used for the inverse PCR
In a first round reaction:
-576 (antisense) 5’-GATCTCTAGTTACCAGAGTCA-3’
+2165 (sense) 5’-CAGAAGAGAGCTTCAGGTTTGGG-3’
In a second round reaction:
-558 (antisense) 5’-GTCACACAACAGACGGGCACAC-3’
+2208 (sense) 5’-TCAGAAGCAGGAGCCGATAGAC-3’
Schematic diagram showing location of the PCR primers used for the inverse
PCR is shown in Supplemental Figure 3.
(2) Primer pairs used for the confirmatory PCR that specifically amplifies
the gene segment spanning the integration site in host genome and the
HIV-1 protease gene
In a first round reaction:
SMC5 F1 (sense) 5’-ACAGGCTAAGCTGGCCATTTGAA-3’
-2589 (antisense) 5’-ACTTTTGGGCCATCCATTCCTGGC-3’
In a second round reaction:
SMC5 F2 (sense) 5’-AAGACGTGCAACCCAAGCAACT-3’
-2556 (antisense) 5’-AGTGCAACCAATCTGAGTCAACA-3’
Schematic diagram showing location of the PCR primers used for the
confirmatory PCR is shown in Supplemental Figure 3.
Supplemental information for Methods
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Site-directed mutagenesis and preparation of virus stocks
To construct an HIV-1 variant containing a stop codon (TGG-to-TAG change) at
codon 42 in the protease gene (PR), site-directed mutagenesis reactions were
carried out using the QuickChange Site-Directed mutagenesis kit (Stratagene) on
a pCR2.1 HIVPR-RT plasmid containing the wild-type RP-RT gene (Apa1-PflM1
fragment of the pNL4.3PFB) as previously described [24]. Primers used for the
point mutagenesis are: sense: 5’GAAATGAATTTGCCAGGAAGATAGAAACCAAAAATGATAGG-3’; antisense: 5’CCTATCATTTTTGGTTTCTATCTTCCTGGCAAATTCATTTC-3’. Transfections
were performed using Transit LT (Invitrogen) as previously described [25].
Approximately 72 hour after transfection, culture supernatants were collected and
cell-free crude supernatants were clarified by low-speed centrifugation (500 x g
for 5 min) and filtered through a 0.2 µm polyvinylidene difluoride membrane filter
(Millipore) to remove cell debris. To pellet HIV-1 virions, 8 mL of the supernatants
were laid on 3 mL 20% sucrose (wt/vol) in 150 mM NaCl-10 mM HEPES, pH 7.4
(Quality Biological, Inc.) and centrifuged at 10,000 x g for 2.5 h at 4°C. The HIV-1
pellet was re-suspended in PBS for HIV-1 infection study or lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail
(Sigma Aldrich) for Western blot analysis. Total amount of protein in each virus
stock was determined using the BCA protein assay reagent (Thomas Scientific).