Correlation between herpes simplex virus type 1 rate of reactivation from latent infection and the number of infected neurons in trigeminal ganglia.

The presence of wild-type herpes simplex virus type 1 (HSV-1) and several latency associated transcript (LAT) region mutants within the trigeminal ganglia (TG) of latently infected mice was examined. A combination of methods including conventional in situ hybridization to detect viral LAT and an in situ DNA polymerase chain reaction (PCR) to detect viral DNA was used. These data show that, for all virus strains in which a comparison was possible, the population of neurons expressing detectable levels of LAT was approximately one-third the total number of viral DNA-containing cells. In addition, in situ PCR analysis revealed that mutants such as 17 delta Sty. 17 delta BstE, and 17 delta S/N, which contain deletions within the LAT locus which do not affect the kinetics of viral reactivation from explanted murine TG, are present in as many neurons as wild-type virus. This was true regardless of the ability to induce accumulation of intact 2.0-kb LAT. On the other hand, mutant 17 delta N/H, which contains a deletion removing the LAT promoter and surrounding genomic region and reactivates slowly from explanted TG, was present in only one-sixth as many neurons as wild-type virus. These data show that detection of mutants unable to synthesize or accumulate 2.0-kb LAT (such as 17 delta N/H) is possible with in situ DNA PCR and that the slow reactivation phenotype of 17 delta N/H correlates with a reduced number of HSV DNA-containing neurons.

In vivo epinephrine reactivation of ocular herpes simplex virus type 1 in the rabbit is correlated to a 370-base-pair region located between the promoter and the 5' end of the 2.0 kilobase latency-associated transcript.

A rabbit ocular model of epinephrine-induced herpes simplex virus type 1 reactivation was employed to study the effect of a deletion in the latency-associated transcript domain. A viral construct derived from 17Syn+, designated 17deltaSty, has a deletion of 370 nucleotides between genomic positions 118880 and 119250. 17deltaSty has been shown to reactivate with wild-type virus kinetics from explants of trigeminal ganglia from latently infected mice. To determine the behavior of this mutant in an in vivo, inducible reactivation system, rabbit corneas were infected with 17Syn+, 17deltaSty, or its rescuant, 17detlaSty-Res. After viral latency was established, transcorneal epinephrine iontophoresis was performed. The rabbits latently infected with 17deltaSty exhibited a significantly reduced ability to undergo adrenergically induced reactivation, i.e., viral shedding in the tears, compared with rabbits infected with either 17Syn+ or 17deltaSty-Res. However, quantitative PCR demonstrated similar numbers of viral genomes in the trigeminal ganglia from rabbits latently infected with all three viruses, and all three viruses reactivated in vitro with wild-type kinetics in an explant cocultivation assay. These studies indicate that the 370-bp region deleted in the 17deltaSty construct plays a role in epinephrine-induced reactivation.

A herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics.

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally. Therefore, to help map the boundaries of the slow reactivation phenotype, a mutant virus with lesions located immediately upstream of the LAT promoter was constructed and called 17 delta S/N. 17 delta S/N contains a 437 nucleotide (nt) deletion 332 nts upstream of the TATAA box of the LAT promoter. In productively infected cells, 17 delta S/N failed to synthesize detectable amounts of the 1.1 and 1.8 kb transcripts which are produced during wild-type infections and are specified by a region just upstream of the LAT promoter. However, 17 delta S/N did produce normal amounts of LAT in tissue culture as well as in neurons derived from latently infected cells, as ascertained by Northern blot and in situ hybridization analysis. Moreover, in latently infected mice, 17 delta S/N established and maintained infection in as many neurons as did wild type virus, as determined by in situ polymerase chain reaction (PCR) to detect viral DNA. Finally, the virus reactivated from TG derived from latently infected mice with kinetics indistinguishable from those of wild-type virus. Therefore, reactivation from latency, in this model system, does not appear to require function from the viral genomic region located immediately upstream of the LAT promoter.

An HSV-1 containing the rat beta-glucuronidase cDNA inserted within the LAT gene is less efficient than the parental strain at establishing a transcriptionally active state during latency in neurons.

The herpes simplex virus vector 17/LAT-RGUSB has previously been shown to express beta-glucuronidase enzyme activity stably in the trigeminal ganglia and brain stems of beta-glucuronidase-deficient mutant mice. However, the number of beta-glucuronidase expressing cells in trigeminal ganglia latently infected with 17/LAT-RGUSB was smaller than expected. Using normal mice for further characterization of 17/LAT-RGUSB latent infection, no appreciable differences were found between the vector and wild-type virus in: (1) their abilities to replicate in acutely infected ganglia; (2) their abilities to reactivate from latently infected ganglia: or (3) the quantities of viral DNA in tissues during the acute or the latent phases of infection. Using a minor LAT (mLAT)-specific probe to detect transcription by in situ hybridization, it was found that the intensity of the signal from individual cells latently-infected with 17/LAT-RGUSB or wild-type virus was similar. However, the vector-infected ganglia had only 20% as many positive cells as in wild-type infection. These data suggest that 17/LAT-RGUSB virus established latency similarly to wild-type virus, but that the LAT-promoter driven gene expression was compromised.

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal ganglia of latently infected mice.

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.

Analysis of a herpes simplex virus type 1 LAT mutant with a deletion between the putative promoter and the 5' end of the 2.0-kilobase transcript.

A herpes simplex virus type 1 strain 17 mutant with a deletion between genomic nucleotides 118880 and 119250 was constructed and called 17 delta Sty. The deletion removes most of a putative secondary LAT promoter (called LAPII) as well as 370 of the first 449 nucleotides of the proposed 8.5-kb transcript believed to be the precursor of 2.0-kb LAT. 17 delta Sty was shown to produce major 2.0-kb LATs in tissue culture. Moreover, trigeminal nerves from latently infected mice contained an intact 1.45- to 2.0-kb LAT as well as the minor LATs which are recognized by probes specific for regions downstream of the 2.0-kb LAT. Finally, 17 delta Sty reactivated with normal kinetics from the trigeminal ganglia of latently infected mice in the explant cocultivation assay and egressed from tissue culture cells as efficiently as wild-type virus. These results clearly show that the region deleted in 17 delta Sty is dispensable for intact 2-kb LAT production, viral egress in tissue culture, and normal reactivation from latently infected neurons in mice.

Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells.

The behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These long-term infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.

An HSV LAT null mutant reactivates slowly from latent infection and makes small plaques on CV-1 monolayers.

A Herpes simplex virus type I (HSV-I) strain 17 mutant deleted between the NotI and HpaI restriction sites of the latency associated transcript (LAT) region has been constructed. The mutant, therefore, contains a deletion of the putative LAT promoter and is called 17N/H. The 17N/H isolate established latent infections in mice nearly as efficiently as its wildtype parent. However, like other LAT null mutants, 17N/H reactivates from explanted ganglia with much slower kinetics than its LAT competent parent. In tissue culture, although 17N/H produces as much virus per cell as its strain 17 parent, it produces small plaques. The small plaque phenotype appears to be due to the inability of the virus to be released from the infected cell into the medium, following low but not high multiplicities of infection (m.o.i.). The mutant was also shown to produce an aberrant LAT homologous transcript of 1.1 kb as well as overproduce an approximately 29,000-Da HSV-specific polypeptide, which is barely detectable in wildtype infected cells. Rescuants of the 17N/H defect were constructed using a 10-kb restriction fragment containing viral sequences spanning the deletion, make large plaques, and have reactivation patterns and infected cell gene product profiles indistinguishable from the 17 parent. This shows that the phenotypes observed in 17N/H are reversed when the deletion, or at most sequences within 5 kb of each side of the deletion, is corrected. The possibilities that the defect in viral egress from infected cell, the small LAT homologous transcript, and the accumulation of the 29,000 Da polypeptide are related to the delayed reactivation kinetics are discussed.