Viral RNA Abundance Research Articles

Productive Human Immunodeficiency Virus Type 1 Infection in Peripheral Blood Predominantly Takes Place in CD4/CD8 Double-Negative T Lymphocytes

Human immunodeficiency virus type 1 (HIV-1) transcription is subject to substantial fluctuation during the viral life cycle. Due to the low frequencies of HIV-1-infected cells, and because latently and productively infected cells collocate in vivo, little quantitative knowledge has been attained about the range of in vivo HIV-1 transcription in peripheral blood mononuclear cells (PBMC). By combining cell sorting, terminal dilution of intact cells, and highly sensitive, patient-specific PCR assays, we divided PBMC obtained from HIV-1-infected patients according to their degree of viral transcription activity and their cellular phenotype. Regardless of a patient's treatment status, the bulk of infected cells exhibited a CD4+ phenotype but transcribed HIV-1 provirus at low levels, presumably insufficient for virion production. Furthermore, the expression of activation markers on the surface of these CD4+ T lymphocytes showed little or no association with enhancement of viral transcription. In contrast, HIV-infected T lymphocytes of a CD4-/CD8- phenotype, occurring exclusively in untreated patients, exhibited elevated viral transcription rates. This cell type harbored a substantial proportion of all HIV RNA+ cells and intracellular viral RNAs and the majority of cell-associated virus particles. In conjunction with the observation that the HIV quasispecies in CD4+ and CD4-)/CD8- T cells were phylogenetically closely related, these findings provide evidence that CD4 expression is downmodulated during the transition to productive infection in vivo. The abundance of viral RNA in CD4-/CD8- T cells from viremic patients and the almost complete absence of viral DNA and RNA in this cell type during antiretroviral treatment identify HIV+ CD4-/CD8 T cells as the major cell type harboring productive infection in peripheral blood.

P21-activated Kinase 1 Is Activated through the Mammalian Target of Rapamycin/p70 S6 Kinase Pathway and Regulates the Replication of Hepatitis C Virus in Human Hepatoma Cells

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.

Positive and Negative Modulation of Viral and Cellular mRNAs by Liver-specific MicroRNA miR-122

microRNAs (miRNAs) are small RNAs that in general down-regulate the intracellular abundance and translation of target mRNAs. We noted that sequestration of liver-specific miR-122 by modified antisense oligonucleotides resulted in a dramatic loss of hepatitis C virus (HCV) RNA in cultured human liver cells. A binding site for miR-122 was predicted to reside close to the 5' end of the viral genome, and its functionality was tested by mutational analyses of the miRNA-binding site in viral RNA, resulting in reduced intracellular viral RNA abundance. Importantly, ectopic expression of miR-122 molecules that contained compensatory mutations restored viral RNA abundance, revealing a genetic interaction between miR-122 and the viral RNA genome. Studies with replication-defective viral RNAs demonstrated that miR-122 affected mRNA abundance by positively modulating RNA replication. In contrast, interaction of miR-122 with the 3'-noncoding region (3'NCR) of the cellular mRNA encoding the cationic amino acid transporter CAT-1 resulted in the down-regulation of CAT-1 protein abundance. These findings provide evidence that a specific miRNA can regulate distinct target mRNAs in both a positive and negative fashion. The positive role of miR-122 in viral replication suggests that this miRNA could be targeted for antiviral therapy.

Transcription of the Herpes Simplex Virus Genome during Productive and Latent Infection

Publisher Summary Herpesviruses are nuclear-replicating, icosahedral, and enveloped DNA viruses that infect members of all groups of vertebrates. Herpesviruses are generally grouped into three divisions—namely, α-herpesviruses, β-herpesviruses, and γ-herpesviruses. Productive replication of all herpesviruses studied to date involves a regulated cascade of viral gene products in which control of viral messenger RNA abundance plays a central role. The plasticity in the structure and organization of herpesvirus genomes is indicative of a basic feature of productive viral replication—herpesvirus genomes are promoter-rich and generally the expression of a given protein is mediated by a specific promoter mapping at that gene. Herpes simplex virus type 1 (HSV 1) is the prototype and best studied representative of the α-herpesvirus group. It is neurotropic and establishes latent infections in sensory neurons. It is characterized by an extremely rapid productive replication cycle and can replicate in a large group of animals, tissues, and cultured cells. Productive infection of a cell by HSV involves a number of stages representing different levels of viral gene expression and interaction of viral gene products with host machinery. Virus entry requires sequential interaction between specific viral membrane glycoproteins and cellular receptors, notably heparan sulfate proteoglycans. The transcriptional events occurring during the programmed interplay between virus and host are shown in the chapter. All of the HSV promoters extensively analyzed are clearly representative of the types of cellular promoters and require cellular transcription factors for activity.

Transcription in mouse embryo cells permissively infected by murine cytomegalovirus.

The sites of transcription and abundance of steady-state cytoplasmic viral RNA in murine cytomegalovirus (Smith strain) infected mouse embryo cells were analyzed. Cloned subgenomic DNA fragments were labeled with 32P and hybridized to filters containing polyadenylated RNA extracted from cells at immediate-early, early, and late times in the infection. The pattern of transcription was distinctive at each time point. Immediate-early transcription occurred primarily at 0.770-0.816 map units. Minor sites of immediate-early transcription were clustered at 0.671-0.861 map units and at the termini of the genome (0.944-0.002 map units). Early transcripts were detected from the entire genome with the exception of a fragment at 0.278-0.305 map units. The site of major immediate-early transcription at 0.770-0.816 map units was represented less abundantly while the concentration of RNA from most other sites of immediate-early transcription was increased at the early time point. The most abundant site of early transcription was at 0.840-0.861 map units. RNA from late in the infection hybridized to all subgenomic fragments. The highest concentration of late RNA transcripts was detected with fragments located at 0.444-0.770 map units. In contrast, late RNA transcripts from both ends of the genome were present at a concentration equal to or lower than that seen at the early time point, and the concentration of late RNA from the major early site (0.840-0.861 map units) was significantly decreased. We also detected uninfected mouse cell RNA with three separate subgenomic EcoRI fragments.

Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169

We used cloned subgenomic DNA fragments of human cytomegalovirus strain AD169 as hybridization reagents to analyze the sites of transcription and abundance of viral RNA in permissively infected human embryonic lung cells. RNA extracted from immediate early, early, middle, and late times in the infection was attached to filters and hybridized with excess cloned subgenomic fragments. Each hybridization was performed with an internal standard to allow quantitation of the RNA concentration and standardization for the variation in probe complexity and specific activity. We found immediate early transcription in the AD169 strain occurring primarily from three regions in the long unique segment at 0.061–0.110, 0.117–0.142, and 0.588–0.616 map units. A low level of transcription was also detected from the long unique segment at 0.230–0.372, 0.419–0.437 and 0.703–0.707 map units and the short unique segment and terminal repeats at 0.00–0.046, 0.776–0.854, and 0.892–1.00 map units. At 8 hr after infection in the presence of de novo protein synthesis, the transcription pattern was changed. Two of the major immediate early sites at 0.061–0.110 and 0.117–0.142 map units were represented less abundantly in the absence of cycloheximide while the site at 0.588–0.616 map units was represented at the same level. The most abundant early RNA was synthesized from the terminal repeat sequences and part of the short unique segment at 0.00–0.046, 0.776–0.822, and 0.892–1.00 map units. Steady-state RNA from the midpoint of the infection hybridized with most regions of the genome. Abundantly transcribed regions included the abundant early sites mentioned above (0.00–0.046, 0.588–0.616, 0.776–0.822, and 0.892–1.00 map units), several new sites in the long unique segment at 0.104–0.110, 0.576–0.588, 0.653–0.664, 0.703–0.707, and 0.766–0.776 map units and the L-S junction fragment at 0.804–0.854 map unit. RNA from late in the infection hybridized to all subgenomic fragments. The sites of de novo RNA synthesis along the genome were determined by hybridizing pulse-labeled RNA to individual cloned subgenomic fragments attached to filters. Transcription from the terminal repeat sequences and the long unique segment at 0.576–0.588 map unit accounted for 40% of the total viral RNA synthesized from 22 to 28 hr after infection. Of the total de novo RNA synthesis from 71 to 77 hr after infection, 4.3% was virus specific and 47% of the total de novo synthesized viral RNA hybridized to the terminal repeat sequences.

Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection.

RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to 8% and 45 to 49% of l and r strands. This assumes that the early class I mRNA (in low concentration late) originates from 8 to 10% and 6 to 10% of l and r strands and that early class II mRNA (in high concentration late) is derived from 2% and 8 to 13% of l and r strands. Mixing experiments indicated that early mRNA is a subset of RNA extracted from polyribosomes late after infection and that late nuclear RNA contains sequences complementary to early l strand class I nRNA. RNA-RNA hybrids were isolated from late mRNA containing sequences from 60% of l and r strands, but it is not known when these were synthesized, and therefore whether complementary RNA transcripts are synthesized late after infection, as they are known to be synthesized early. These results demonstrate that portions of the genome are transcribed into RNA sequences that remain confined to the nucleus and are not exported to polyribosomes as mRNA.