Herpes simplex virus Research

Temporal Patterns of HSV-2 Transcripts

HSV-1 and HSV-2 (HHV1 and HHV2) are very closely related by sequence, and while both viruses are medically important, to date the bulk of basic research on the basic virology and molecular biology of HSV including patterns of viral gene expression during replication and latency has been carried out with HSV-1.

Despite their high degree of genomic identity (>80%), the significantly different pathology in humans exhibited by these two viruses is reflected in differences in the behavior of the virus in animal systems. An important problem in herpes virology is to understand the molecular basis leading to the different pathologies between HSV-1 and HSV-2. Differences in viral functions as well as in the cellular functions affected by each type of virus could, at least partially, explain these different pathologies.

For instance, as shown by Herold and colleagues, while in HSV-1 glycoprotein C is the major viral function responsible for virus attachment to cells and glycoprotein B mediates penetration, in HSV-2 glycoprotein B is the major protein involved both in binding and penetration. Also, Leib and coworkers have shown the viral host shut off activity is much stronger in HSV-2 than in HSV-1.

Cellular functions are also affected differently in HSV-1 and HSV-2 infections. While a full catalogue of such differences will require detailed analysis of global transcription patterns following approaches similar to those outlined in this site, it has been well established that the levels of specific transcription factors such as c-Jun, NF-kB , and c-fos are differentially affected by infections by HSV-1 and HSV-2. As summarized by Dolan and colleagues, the general transcription map of HSV-2 is very similar to that of HSV-1, but until the application of microarrays, the kinetic characteristics of many HSV-2 genes have not been described, and it is important to determine the kinetic characteristic of HSV-2 genes and compare them with their counterparts in HSV-1 as a basis for a fuller understanding of the differences between HSV-1 and HSV-2.

We have used the approach described for the HSV-1 chip to design an oligonucleotide-based array specific for HSV-2. For the HSV-2 chip, a total of 97 probes were synthesized using information obtained for the HG-52 strain in Genbank, and printed on chips. These were then tested by hybridization to dye-labeled nick-translated cloned HSV-2 fragments covering the entire genome. The 75 probes, which showed the highest hybridization values and little or no cross hybridization with non-homologous DNA fragments were used for statistical analysis of transcript abundance.

To examine immediate early transcription, we isolated RNA from both HSV-1 and HSV-2 infected human fibroblasts four hours after infection in the presence of 100 mg/ml cycloheximide, and synthesized dye-labeled cDNA. A number of HSV-2 transcripts can be detected with some efficiency using homologous HSV-1 probes in northern blots; therefore, we hybridized the dye-labeled cDNA to HSV-1 chips.

The Expression of HSV-2 Immediate Early and Selected Early Transcripts
in the Presence of 100 mg/ml Cycloheximide

Gene	HSV-1		HSV-2
	Median	SD	Median	SD
ICP27	27100	2100	39900	11600
ICP0	60900	38300	47200	4100
ICP4	48900	11100	86800	30100
ICP22	47100	19600	42800	10400
ICP47	36000	25000	36200	18200
UL39	4500	5000	6300	300
UL23	3000	2100	1100	1000
UL50	2200	2200	2600	2200

The four putative HSV-2 immediate early transcripts, ICP4, 0, 22, 27, and 47 all hybridized efficiently to HSV-1 chips. Under these conditions, the transcript encoding the large subunit of ribonucleotide reductase hybridized to levels only insignificantly higher than two early transcripts (U23-tk and U50-dUTPase) included for comparison. This suggests that in HSV-2, like HSV-1, the UL39 transcript is abundant under conditions favoring immediate-early expression.

We compared the patterns of expression of those HSV-2 transcripts whose probes were essentially equivalent in position and specificity to their HSV-1 homologues under conditions of PAA blockage of viral DNA replication. For these experiments, RNA was isolated at 6 hr after infection in the presence of 200 mg/ml PAA to inhibit DNA replication. Hybridization of HSV-2 RNA to the HSV-2 chip for both untreated and drug-treated samples at 6 hr pi are shown below. Transcripts are grouped by the kinetic class of the representative HSV-1 example, and it is evident that there is a general correspondence in the overall patterns of abundance between the two virus types. This confirms the general correspondence of early kinetics between HSV-1 and HSV-2 transcripts, but notable exceptions can be seen and are being examined more detail.
transcript abundance

For analysis of specific differences between HSV-1 and HSV-2 transcript abundance patterns human fibroblasts were infected with HSV-2 and RNA isolated at 2, 8, and 16 hours following infection. Such RNA was then used as a template for synthesis of dye-labeled cDNA and this was hybridized to HSV-2 chips. The median, normalized hybridization values for the various transcripts demonstrate a general correspondence in kinetic class between HSV-1 and HSV-2, but detailed comparisons of representative immediate-early, early, and late transcripts reveal some notable differences in timing and relative abundance. Further, more extensive experimental analysis including more detailed time variance studies, analysis of the effect of metabolic inhibitors, and more careful analysis of individual promoter elements will be required to fully assess the significance of the differences noted. It is to be expected that such differences will have an important role in differences in the patterns of viral pathogenesis.