E K Wagner

Global Analysis of Herpes Simplex Virus Type 1 Transcription Using an Oligonucleotide-Based DNA Microarray

ABSTRACT More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early α27 (UL54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the α27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.

Individual HSV Transcripts

An increasingly complete picture of the phenomenology of animal vims gene expression during productive infection is at hand. In the case of herpesviruses [particularly herpes simplex virus type 1 (HSV-1)], this picture is dependent on the revolution in molecular biology resulting from restriction enzyme analysis of viral DNA and, more recently, from the use of recombinant DNA technology for the construction and analysis of fine probes of HSV-1 DNA transcription. These powerful techniques, along with techniques available from the parallel revolution in immunology and assays of biological activities, suggest that a detailed mechanistic description of the intricacies of HSV replication is technically feasible.

Immediate-Early Expression of the Herpes Simplex Virus Type 1 ICP27 Transcript Is Not Critical for Efficient Replication In Vitro or In Vivo

ABSTRACT We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

Wide Variations in Herpes Simplex Virus Type 1 Inoculum Dose and Latency-Associated Transcript Expression Phenotype Do Not Alter the Establishment of Latency in the Rabbit Eye Model

ABSTRACT The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LATs mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17ΔPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.

Design of a herpes simplex virus type 2 long oligonucleotide-based microarray: global analysis of HSV-2 transcript abundance during productive infection.

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains.

The Herpes Simplex Type 1 Virus Latency Gene

The ability of herpesviruses to establish, maintain, and reactivate from latent infections is a hallmark of the group. Latent phase gene expression is under different control than is herpesvirus lytic phase infection. Recent experimental analysis of herpes simplex virus latency indicates that the latent phase is characterized by the expression of a single transcript unit (the latency-associated transcription unit, LAT). Transcripts expressed from this LAT are complexly processed to yield a stable intron and a number of, as yet, not fully characterized polyadenylated forms. One or more of these processed transcripts play an important role in the reactivation process, but the biological mechanism underlying this role is not understood.

Herpes simplex virus type 1 shows multiple interactions with sulfonated compounds at binding, penetration, and cell-to-cell passage

Herpes simplex virus type 1 (HSV-1) uses multicomponent mechanisms for binding, penetration, and cell-to-cell passage. These processes are affected by polysulfonated compounds. In this paper we have addressed the question of whether the same or different interactions of HSV-1 with polysulfonated compounds are involved in binding, penetration, and passage. For this, we have compared the inhibitory dose–response for a series of polysulfonated and cationic compounds known to block HSV-1 infections. These comparisons were done at the level of binding, penetration, and cell-to-cell passage. Variations in the parameters of the dose–response curves––IC50 and Hill coefficients (nH)––are consistent with HSV-1 having multiple interactions with sulfonated cellular components in all these processes. Some of the interactions seem to be common to the three processes, while others are particular for each one.