Transcription of herpes simplex virus genes in vivo: overlap of a late promoter with the 3' end of the early thymidine kinase gene

We compared the rates of synthesis of herpes simplex virus type 1 glycoproteins C and D and quantitated the accumulation of translatable mRNA for each glycoprotein at various times after infection. The rate of synthesis of gD increased sharply early in the infection, peaked by 4 to 6 h after infection, and declined late in the infection. In contrast, the rate of synthesis of gC increased steadily until at least 15 h after infection. The levels of mRNA for both of these glycoproteins, as detected by hybridization and by translation in vitro, continued to increase until at least 15 or 16 h after infection. Synthesis of both gC and gD and their respective mRNAs was found to be sensitive to inhibition of viral DNA replication with phosphonoacetic acid. The finding that reduced amounts of gD were synthesized late in the replicative cycle, whereas gD mRNA continued to accumulate in the cytoplasm, argues that the synthesis of gD is regulated, in part, at the level of translation.

supporting

confidence: 58%

Evidence for translational regulation of herpes simplex virus type 1 gD expression

Johnson¹,

Spear²

1984

“…1C, right). Another L transcript, that of gH, is known to be colinear with a transcript arising from the E gene tk, located upstream of gH (17,19,46). To determine the level of promoter-specific RNA from gH, we asked if potential contributions to the QRPCR measurement from upstream run-on transcripts could be subtracted.…”

Section: Resultsmentioning

confidence: 99%

Accumulation of Viral Transcripts and DNA during Establishment of Latency by Herpes Simplex Virus

Kramer¹,

Chen²,

Knipe

et al. 1998

Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.

“…With lacZ upstream of the albumin enhancer/promoter, it is very unlikely that read-through transcription from the TK promoter could occur. The 3Ј untranslated region of TK, downstream of the ICP4 transgene, overlaps the 5Ј promoter region of gH (65), which should have no impact on the regulation of ICP4 expression. pTK⌬L-ALI4 was cotransfected with d120 DNA into E5 (ICP4 ϩ ) cells, and the resulting recombinant viruses were thrice plaque purified on E5 cells.…”

Section: Resultsmentioning

confidence: 99%

“…The second chimeric transgene, with the Escherichia coli lacZ gene under control of the HSV-1 TK promoter, was used to easily screen for recombinant viruses and identify cells containing replicating virus. TK is an E gene, and therefore lacZ expression should be dependent on synthesis of ICP4 protein (11,16,57,65,78). The chimeric transgenes (albumin enhancer/promoter-ICP4, TK promoter-lacZ) were inserted into the HSV-1 TK gene (UL23), resulting in a 0.5-kb deletion of the TK gene and inactivation of the UL24 gene (31).…”

mentioning

confidence: 99%

Transcriptional targeting of herpes simplex virus for cell-specific replication

Miyatake

Iyer

Martuza

et al. 1997

Tissue-or cell-specific targeting of vectors is critical to the success of gene therapy. We describe a novel approach to virus-mediated gene therapy, where viral replication and associated cytotoxicity are limited to a specific cell type by the regulated expression of an essential immediate-early viral gene product. This is illustrated with a herpes simplex virus type 1 (HSV-1) vector (G92A) whose growth is restricted to albuminexpressing cells. G92A was constructed by inserting an albumin enhancer/promoter-ICP4 transgene into the thymidine kinase gene of mutant HSV-1 d120, deleted for both copies of the ICP4 gene. This vector also contains the Escherichia coli lacZ gene under control of the thymidine kinase promoter, a viral early promoter, to permit easy detection of infected cells containing replicating vector. In the adult, albumin is expressed uniquely in the liver and in hepatocellular carcinoma and is transcriptionally regulated. The plaquing efficiency of G92A is >10 3 times higher on human hepatoma cells than on non-albumin-expressing human cells. The growth kinetics of G92A in albumin-expressing cells is delayed compared with that of wild-type HSV-1, likely due to aberrant expression of ICP4 protein. Cells undergoing a productive infection expressed detectable levels of ICP4 protein, as well as the reporter gene product ␤-galactosidase. Confining a productive, cytotoxic viral infection to a specific cell type should be useful for tumor therapy and the ablation of specific cell types for the generation of animal models of disease.