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Differential Gene Expression

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Differential Gene Expression JOURNAL OF VIROLOGY, Sept. 1989, p. 3708-3713 Vol. 63, No. 9 0022-538X/89/093708-06$02.00/0 Copyright © 1989, American Society for Microbiology Temporal Aspects of DNA and RNA Synthesis during Human Immunodeficiency Virus Infection: Evidence for Differential Gene Expression SUNYOUNG KIM,"2 RANDAL BYRN,3 JEROME GROOPMAN,3 AND DAVID BALTIMORE12* Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 021421*; Department ofBiology, Massachusetts Institlute of Technology, Cambr-idge, Massac hulsetts 021392; and New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 021153 Received 13 March 1989/Accepted 24 May 1989 The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes. Synthesis of DNA from the genomic RNA template and tant was mixed with H9 cells, a human CD4+ lymphocyte transcription of viral RNA from the viral DNA are early line (18), 2 to 3 days posttransfection. Approximately 70 to steps in the retroviral life cycle. Although these steps have 90% of these H9 cells were infected after 7 to 14 days. At this been well studied in simpler retroviruses (for a review, see time, a high concentration of virus was prepared by the reference 27), the sequential pathways of DNA and RNA shaking method (29) and then filtered through the 0.45- synthesis in human immunodeficiency virus (HIV) have not .zm-pore-size filter. To give the highest rate of initial infec- been investigated. It is clear that these steps are not uniform tion, virus was used immediately for infection of fresh H9 among retroviruses. The diversity in aspects of DNA syn- cells. H9 cells were infected with HIV type 1 (HIV-1) W13 as thesis is exemplified by spleen necrosis virus (10) and follows. Cells were treated with polybrene at 37°C for 1 h, Rous-associated virus (30), in which a high copy number of collected by centrifugation, and exposed to virus at 37°C for unintegrated linear DNA has been correlated with cell death, 1.5 h. They were then washed with medium and divided and by visna virus, which may not integrate its DNA into the among an appropriate number of culture flasks. For most of genome of host cells, at least in tissue culture (7). It has also the experiments described here, each bottle initially con- been hypothesized that for HIV, unintegrated viral DNA tained approximately 107 cells. At each time point, samples may be used as a template for transcription and that the high were taken for reverse transcriptase assay (17), indirect copy number of unintegrated viral DNA may result in cell immunofluorescence assay (9), viable cell count, and prep- death (15, 23). To examine such possibilities, we have aration of low-molecular-weight DNA. (Times indicated in studied the timing and quantity of viral DNA synthesis the figures include 1.5 h of virus exposure time.) following HIV infection. In addition, the complex nature of Primary T cells were prepared as described elsewhere (6, the genome-nine genes in its 9.2 kilobases (kb)-and the with 2 of ml for 2 action of the two best-characterized trans-acting regulatory 32) and treated pg phytohemagglutinin per genes, tat and rev (for recent articles, see references 4, 5, 11, to 3 days before infection. 12, 19, and 20), indicate that HIV might have an efficient way DNA blot hybridization. Low-molecular-weight DNA was of regulating its own gene expression during its infection prepared from infected cells by Hirt extraction (8) and cycle. Therefore, we have studied the species of viral RNA subjected to Southern blot analysis (24). For quantitative present at different times after infection. We find that viral measurement of the copy number of unintegrated HIV DNA synthesis is similar to that of standard retroviruses but DNA, the total DNA fraction, including high- and low- that there is temporal control of the species of viral mRNA molecular-weight DNAs, was prepared. Five million cells that accumulate. were treated with 1% sodium dodecyl sulfate and 400 p.g of proteinase K per ml, and 1/60 of these DNA preparations MATERIALS AND METHODS was directly subjected to Southern blot analysis. It has to be noted that this procedure consistently yields a lower ratio of Virus and infection. To produce virus, COS cells were circular to linear DNA than the Hirt procedure. The DNA transfected with the plasmid pWI3. pWI3 is essentially the probe used for hybridization was the Sacd fragments of same as RIP7 (14) except that the former contains the full nef pWI3, which includes most of the HIV genomic DNA. The coding region derived from pCV-3 (1). The culture superna- intensities of HIV-specific DNA bands were determined by UltroScan Laser Densitometer (LKB 2202). X-ray film was * Corresponding author. preflashed before exposure to the filter. 3708 VOL. 63, 1989 DNA AND RNA SYNTHESIS IN HIV INFECTION 3709 LQ 4 4~ 8 12 in242? v 33 4s368 83 0 15U Ch -x -L -c - X L 0kIJ -C Lu I B 3 3 5 12 2-3 24 28 33 44 52 h6 11kI. v4p52S3L Lut-C1.) LU. Z (- 5 '2 22 31 HOURS FIG. 1. One-step growth curve of HIV. IF, Change in the percentage of immunofluorescent-positive cells during the course of infection; RT, change in reverse transcriptase activity in cell culture supernatant. FIG. 2. Hybridization analysis of HIV-specific low-molecular- RNA blot hybridization. Total cellular and cytoplasmic weight DNA. (A) Total low-molecular-weight DNAs. Short (a) and RNAs were prepared by the guanidine thiocyanate-cesium long (b) exposures of the same autoradiogram are shown. (B) chloride method (2). For cytoplasmic RNA, the cytoplasmic Cytoplasmic (a) and nuclear (b) low-molecular-weight DNAs. (C) Low-molecular-weight DNA from infected primary T cells. Short (a) fraction was prepared with the detergent Nonidet P-40 and The DNA probe and long (b) exposures of the same autoradiogram are shown. then mixed with guanidine thiocyanate. Numbers at the top of lanes are hours after the infection was started. used for hybridization was the 511-base-pair BglII fragment Lane Ch shows a DNA sample from a chronically infected culture. of pWI3, which includes the polyadenylation signal se- L, C, and X, Linear, circular, and nicked circular DNAs, respec- quence. tively. H9 cells or primary T cells (enriched for CD4+ cells) were infected with HIV-1 W13, and low-molecular-weight DNA was RESULTS prepared by Hirt extraction (8) and subjected to Southern blot analysis (24). Cytoplasmic and nuclear fractions were prepared from Establishment of a one-step growth of HIV. Investigating the same cells by using the detergent Nonidet P-40 and then the kinetics of DNA and RNA synthesis required the estab- subjected to Hirt extraction. Because of limits in Hirt extraction, it is difficult to quantitatively compare the amounts of linear DNA in a for HIV. lishment of single-cycle growth condition To the cytoplasm and the nucleus. achieve this, we used a procedure for producing reliably high-titer HIV-1 stocks from a homogeneous, cloned viral DNA, thus allowing synchronous infection of 10 to 20% of (designated L, C, and X, respectively [Fig. 2]) by restriction H9 or primary T cells. When H9 cultures were analyzed at enzyme digestion as well as by their sizes (data not shown). various times after infection, a significant number of immu- HIV-specific linear DNA could be detected 4 h after nofluorescence-positive cells were first observed 28 to 32 h initiation of infection, while the circular form was first postinfection, coinciding with an exponential increase in detectable between 8 and 12 h (Fig. 2A; note that two reverse transcriptase activity in cell culture supernatants exposures of the same gel are presented). The ratio of (Fig. 1). Immunofluorescence indicated the expression of circular to linear DNA increased as the viral infection viral structural gene products, while reverse transcriptase progressed, but linear DNA remained the dominant form measured the active release of virus particles, suggesting the throughout the one-step growth cycle. After the first cycle of end of a one-step growth cycle. This interpretation is rein- infection was completed, between 28 and 32 h postinfection, forced by the DNA and RNA analysis described below. the amount of viral DNA increased greatly as the infection Therefore, by making observations during the first 32 h after spread through the culture (Fig. 2A). infection, we could approximate one-step growth conditions. To localize the different viral DNA species, cytoplasmic DNA synthesis during a one-step growth of HIV. To study and nuclear fractions were prepared with the detergent temporal aspects of viral DNA synthesis, low-molecular- Nonidet P-40 and low-molecular-weight DNA was made weight DNA was prepared from infected cells by Hirt from each fraction.
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