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. 2014 Feb 5;9(2):e87914.
doi: 10.1371/journal.pone.0087914. eCollection 2014.

Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART

Jesse D Deere  1 , Robert C Kauffman  1 , Elda Cannavo  1 , Joanne Higgins  1 , Andradi Villalobos  1 , Lourdes Adamson  1 , Raymond F Schinazi  2 , Paul A Luciw  3 , Thomas W North  4
Affiliations

Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART

Jesse D Deere et al. PLoS One. .

"VSports app下载" Abstract

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus VSports手机版. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load. .

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Conflict of interest statement (V体育ios版)

Competing Interests: The authors have declared that no competing interests exist.

V体育平台登录 - Figures

Figure 1
Figure 1. Map of the MS mRNA splice site.
Genome map depicting the MS transcript region of RT-SHIV. There are two open reading frames on the MS transcript, encoding the Tat and Rev proteins. The Envelope protein (Env) is also encoded through this region. A portion of full-length viral sequence surrounding the splice junction is displayed (blue bar with sequence below). The splice donor and the alternative acceptor sites for the MS transcript are indicated with a red asterisk. A splicing event with splice acceptor 1 would result in a viral transcript containing the CAG sequence, while an event with splice acceptor 2 would result in a transcript without the CAG sequence.
Figure 2
Figure 2. Validation of TaqMan PCR for MS mRNA, multiplex preamplification PCR, and distribution of GAPDH RNA in macaque tissues.
A) Vectors containing a portion of the MS transcript with or without the CAG nucleotides at the coding exon-1 and coding exon-2 splice junction were used as standards for real-time TaqMan PCR. The plasmids were quantified by A260 spectrophotometric readings and serially diluted for quantification by TaqMan PCR. Data shown are the average threshold cycle (Ct) +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis used to obtain line equations for calculating the amplification efficiencies and for the quantification of MS mRNA in samples. B) cDNA was generated from total RNA from RT-SHIV-infected CEMx174 cells isolated 48 hours post-infection using random hexamers primers. Multiplex preamplification PCR was performed for viral MS mRNA and gag RNA in addition to the cellular housekeeping gene GAPDH RNA. Individual reactions were removed from the cycler every five cycles, followed by quantification of the three transcripts by TaqMan PCR to determine whether the preamplification step was linear. Data shown are the average +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis. C) Total RNA was isolated from tissues and quantified by two-step TaqMan RT-PCR. Total cDNA was preamplified for 25 cycles in multiplex PCR before TaqMan PCR quantification. Cellular GAPDH RNA was measured in these six tissues from all nine macaques in order to determine whether it could serve as a standard. The solid symbols indicate samples from animals that received only HAART while the open symbols indicate samples from animals that received HAART plus induction therapy. The horizontal lines represent the averages with 95% confidence intervals.
Figure 3
Figure 3. MS mRNA and gag RNA levels correlate with plasma virus loads in macaques.
The correlations were determined between plasma virus loads and levels of the MS transcript (red circles) and gag RNA (blue squares) in tissues from infected macaques. The solid symbols indicate samples from animals that received only HAART while the open symbols indicate samples from animals that received HAART plus induction therapy. All nine animals from the study are represented in the figure. Both axes represent log RNA levels and the dashed line indicates the limit of detection.
Figure 4
Figure 4. Ratio of MS mRNA to gag RNA in gastrointestinal tissues.
The ratios of the MS transcript to gag RNA in duodenum, jejunum, and colon were calculated in order to determine whether there was an abundance of MS mRNA in tissues from macaques treated with HAART. Bars indicate average with standard error of the mean. Horizontal line indicates the results of an unpaired two-tailed student t-test.
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