Dataset Information

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

ABSTRACT: Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8. 4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8. 0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8. 0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8' and 8. Its synthesis was inhibited by cycloheximide but not by cytosine beta-D-arabinofuranoside, which suggests that the 2. 0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

SUBMITTER: Jin L

PROVIDER: S-EPMC113025 | biostudies-literature | 1999 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Jin L L Scherba G G

Journal of virology 19991201 12

Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not be ...[more]

PMID: 10559288

Similar Datasets

Project description:UnlabelledThe alphaherpesvirus pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only the transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within the LAT, suggesting a role in establishment and/or maintenance of latency. We generated a mutant (M) PrV deleted of nine miRNA genes which displayed properties that were almost identical to those of the parental PrV wild type (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT or M virus or were mock infected. Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days postinfection, trigeminal ganglia were excised and profiled by deep sequencing and quantitative RT-PCR. Latency was established in all infected animals without evidence of viral reactivation, demonstrating that miRNAs are not essential for this process. Lower levels of the large latency transcript (LLT) were found in ganglia infected by M PrV than in those infected by WT PrV. All PrV miRNAs were expressed, with highest expression observed for prv-miR-LLT1, prv-miR-LLT2 (in WT ganglia), and prv-miR-LLT10 (in both WT and M ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M, and control ganglia. Both viruses triggered a strong host immune response, but in M ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and the migration of dendritic cells, were impaired in M ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state.ImportanceThis study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV, the only remarkable differences are found in the pattern of host response. This indicates that, as in herpes simplex virus, LAT functions as an immune evasion locus.

Project description:Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

Project description:BACKGROUND: Herpesvirus genes are classified into distinct kinetic groups on the basis of their expression dynamics during lytic growth of the virus in cultured cells at a high, typically 10 plaque-forming units/cell multiplicity of infection (MOI). It has been shown that both the host response and the success of a pathogen are dependent on the quantity of particles infecting an organism. This work is a continuation of an earlier study 1, in which we characterized the overall expression of PRV genes following low-MOI infection. In the present study, we have addressed the question of whether viral gene expressions are dependent on the multiplicity of infection by comparing gene expressions under low and high-MOI conditions. RESULTS: In the present study, using a real-time RT-PCR assay, we address the question of whether the expression properties of the pseudorabies virus (PRV) genes are dependent on the number of virion particles infecting a single cell in a culture. Our analysis revealed a significant dependence of the gene expression on the MOI in most of these genes. Specifically, we found that most of the examined viral genes were expressed at a lower level at a low MOI (0.1) than at a high MOI (10) experiment in the early stage of infection; however, this trend reversed by six hour post-infection in more than half of the genes. Furthermore, in the high-MOI infection, several PRV genes substantially declined within the 4 to 6-h infection period, which was not the case in the low-MOI infection. In the low-MOI infection, the level of antisense transcript (AST), transcribed from the antiparallel DNA strand of the immediate-early 180 (ie180) gene, was comparable to that of ie180 mRNA, while in the high-MOI experiment (despite the 10 times higher copy number of the viral genome in the infected cells) the amount of AST dropped by more than two log values at the early phase of infection. Furthermore, our analysis suggests that adjacent PRV genes are under a common regulation. This is the first report on the effect of the multiplicity of infection on genome-wide gene expression of large DNA viruses, including herpesviruses. CONCLUSION: Our results show a strong dependence of the global expression of PRV genes on the MOI. Furthermore, our data indicate a strong interrelation between the expressions of ie180 mRNA and AST, which determines the expression properties of the herpesvirus genome and possibly the replication strategy (lytic or latent infection) of the virus in certain cell types.

Dataset Information

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Publications

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets