Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns & Allen Research Institute, Davis Bldg Room 5072, 8700 Beverly Blvd, Los Angeles, CA 90048, USA1
Department of Ophthalmology, UCLA School of Medicine, Los Angeles, CA, USA2
Author for correspondence: Steven Wechsler (at Ophthalmology Research Laboratories). Fax +1 310 423 0225. e-mail Wechsler{at}csmc.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Latency-associated transcript (LAT) is the only viral gene abundantly transcribed during HSV-1 neuronal latency. In the rabbit ocular model LAT null mutants consistently have reduced in vivo reactivation (Bloom et al., 1994 ; Perng et al., 1994
, 1996
; Trousdale et al., 1991
). In contrast, in the mouse model of explant-induced reactivation (EIR) of TG, the effect of LAT on reactivation is less clear. At least three reports indicate that mutants unable to transcribe LAT reactivate similarly to LAT+ virus in the mouse (Cook et al., 1991
; Deshmane et al., 1993
; Javier et al., 1988
). Several other mutants thought at the time to disrupt LAT function also did not alter the EIR phenotype in mice (Block et al., 1990
; Ho & Mocarski, 1989
; Izumi et al., 1989
; Junejo & Brown, 1995
; Natarajan et al., 1991
). In contrast, in other reports LAT mutants had significantly reduced reactivation in the mouse (Block et al., 1993
; Devi-Rao et al., 1994
; Leib et al., 1989
; Sawtell & Thompson, 1992
; Steiner et al., 1989
). To add to the confusion, in at least two instances LAT mutants with normal EIR phenotypes in mice (Maggioncalda et al., 1994
, 1996
) were later shown by the same investigators to have significantly reduced reactivation in the rabbit (Hill et al., 1996
, 1997
).
LAT can enhance the efficiency of establishing latency (Perng et al., 2000a , b
; Thompson & Sawtell, 1997
), which may account for some of the effect of LAT on reactivation. Note that in this report we use the term reactivation and explant-induced reactivation (EIR) as a phenotype, much like blue eyes. Thus, even if the ability of LAT to increase EIR was completely due to the ability of LAT to enhance the efficiency of establishing latency (Perng et al., 2000a
, b
; Thompson & Sawtell, 1997
), we would still say that LAT enhances reactivation. For clarity the term reactivation phenotype or EIR phenotype will be used below when appropriate.
To investigate the above discrepancies regarding LAT in the mouse EIR phenotype, we infected BALB/c and Swiss Webster mice with LAT- or LAT+ virus. We report here that in BALB/c mice no significant differences were seen in the EIR phenotype. In contrast, in Swiss Webster mice the EIR phenotype of the LAT- virus was significantly decreased.
dLAT2903, the LAT- virus used here, contains a 1·8 kb deletion in both copies of LAT (Perng et al., 1994 ). The deletion removes the primary TATA box-based LAT promoter and a second putative promoter located just prior to the stable 2 kb LAT region. The deletion also encompasses the first 1·67 kb of the primary 8·3 kb LAT and almost 1 kb of the 2 kb LAT. This mutant makes no detectable LAT transcripts, yet replicates in tissue culture, rabbit eyes and rabbit TG in a manner indistinguishable from the parental McKrae virus or marker-rescued dLAT2903R (Perng et al., 1994
). In rabbits dLAT2903 has a reduced reactivation phenotype (Perng et al., 1994
).
BALB/c mice and Swiss Webster mice were ocularly infected with 102, 103, 104, 105 or 106 p.f.u./eye of LAT- or LAT+ virus, without corneal scarification. Because of expected differences in survival rates at different infectious doses, five mice/group were infected with the 102 and 103 p.f.u./eye doses, 10 mice/group were infected with the 104 p.f.u./eye dose, and 25 mice/group were infected with the 105 and 106 p.f.u./eye doses. TG were removed 30 days post-infection for determination of the kinetics of EIR.
Since more Swiss Webster mice than BALB/c mice survived (see below), some of the TG from the Swiss Webster mouse groups were randomly eliminated from the study such that the numbers of TG in each Swiss Webster mouse group were the same as the corresponding BALB/c mouse group. Thus any apparent differences in EIR between the BALB/c and Swiss Webster mice should not be due to unequal statistical power.
The cumulative percentages of TG that reactivated during the 18 day observation period are shown in Fig. 1(AH
). No significant reactivation was detected in either BALB/c or Swiss Webster mice following infection at 102 p.f.u./eye (Fig. 1I
, J
). In BALB/c mice infected with 103 (Fig. 1A
) and 106 (Fig. 1D
) p.f.u./eye, reactivation of the LAT- and LAT+ viruses was virtually identical. At 104 and 105 p.f.u./eye (Fig. 1B
, C
), the reactivation of LAT- appeared slightly reduced compared to LAT+ in the BALB/c mice, but the differences were not significant. Thus, as summarized in Fig. 1(I
), at all doses there was no significant difference in EIR of the LAT- and LAT+ viruses from TG of BALB/c mice (P>0·1).
|
BALB/c mice and Swiss Webster mice were ocularly infected with 105 or 106 p.f.u./eye of LAT- or LAT+ virus. Tears were collected at various times post-infection and virus was quantified by standard plaque assays as previously described (Perng et al., 1994 ) (Fig. 2
). At 105 p.f.u./eye the peak titres for these viruses were similar in BALB/c mice (Fig. 2A
, day 5, P=0·42) and in Swiss Webster mice (Fig. 2B
, day 5 LAT+ compared to day 3 LAT- P=0·20). At 106 p.f.u./eye, both viruses had similar replication kinetics and peak titres in the eyes of BALB/c mice (Fig. 2C
; P=0·1 and P=0·4 for day 5 and 7 respectively) and Swiss Webster mice (Fig. 2D
; P=0·11 for day 5). There was also no significant difference in peak virus titres of LAT- and LAT+ in TG of BALB/c or Swiss Webster mice (not shown). These results are similar to our previous findings with these viruses in rabbits (Perng et al., 1994
). Although peak virus titres were similar in the eyes of both mouse strains, the titres appeared to remain high for an extended time in eyes of BALB/c mice. This may be related to the decreased survival of BALB/c mice compared to Swiss Webster mice shown below.
|
|
To determine if the findings presented here, i.e. that LAT was required for wild-type levels of EIR from Swiss Webster mouse TG but not BALB/c TG, are consistent with previous reports, we re-examined the literature. We found five publications in which apparently well-defined, molecularly constructed, LAT null mutants (i.e. mutants that were constructed such that they are deleted for the LAT promoter, with or without deletion of adjacent regions) were reported to have reduced or delayed EIR phenotypes in mice (Block et al., 1993 ; Devi-Rao et al., 1994
; Leib et al., 1989
; Sawtell & Thompson, 1992
; Steiner et al., 1989
). In three of these papers (Devi-Rao et al., 1994
; Leib et al., 1989
; Sawtell & Thompson, 1992
) the studies were done in Swiss Webster mice or CD-1 mice, which, like Swiss Webster mice, are an outbred strain. These reports are therefore consistent with the results reported here. The remaining two studies were done in BALB/c mice (Block et al., 1993
; Steiner et al., 1989
). However, the mutants used in both of these studies had unexpected genetic defects in addition to the LAT deletion. In one case the LAT- virus made small plaques and grew poorly (Block et al., 1993
). Neither of these phenotypes have been reported in any other defined LAT- virus. In the other case (Steiner et al., 1989
), the LAT- virus was later shown to be defective for gC also (Wroblewska et al., 1991
).
Of the three reports suggesting that LAT- viruses have EIR phenotypes similar to that of LAT+ virus, one was done in BALB/c mice (Deshmane et al., 1993 ), and this is consistent with the results we reported here. The other two were done in Swiss Webster mice. However, both of these studies used the LAT- mutant x10-13 (Cook et al., 1991
; Javier et al., 1988
). This virus was fortuitously derived from an HSV-1xHSV-2 intertypic recombinant (Javier et al., 1987
) during the course of marker rescue experiments in mouse brain following intracranial inoculation (Javier et al., 1988
). Thus, in addition to containing HSV-2 sequences, x10-13 may contain numerous unknown alterations. In addition, in one of the x10-13 studies, no marker-rescued LAT+ virus was used, EIR was not studied kinetically, and the EIR was done with dorsal root ganglia, not TG (Javier et al., 1988
). In the other study, the number of mice was very small, only six per group (Cook et al., 1991
). This study reported no statistical significance between explant reactivation of the LAT- (x10-13) virus and the LAT+ virus, with a P value of 0·09. It is possible that statistical significance would have been reached had a larger number of mice been used. Thus, consistent with the results reported here, all of the truly well-defined LAT- viruses that were reported to have decreased EIR phenotypes in mice were studied in either CD-1 or Swiss Webster mice, and all of the truly well-defined LAT- viruses reported to have normal EIR phenotypes in mice were studied in BALB/c mice.
The results reported here, combined with the above survey of the literature, suggest that in contrast to other mouse strains, rabbits and presumably humans, in BALB/c mice the presence or absence of LAT has little effect on the EIR phenotype of HSV-1 from TG. The genetic difference in BALB/c mice that is responsible for this remains to be determined.
In summary, the results reported here suggest that in Swiss Webster mice it is easy to detect the effect of LAT on the EIR phenotype, while in BALB/c mice such an effect is difficult to observe and, if present, may be highly dependent on initial infectious dose of the viruses.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Block, T. M., Deshmane, S., Masonis, J., Maggioncalda, J., Valyi-Nagi, T. & Fraser, N. W. (1993). An HSV LAT null mutant reactivates slowly from latent infection and makes small plaques on CV-1 monolayers. Virology 192, 618-630.[Medline]
Bloom, D. C., Devi-Rao, G. B., Hill, J. M., Stevens, J. G. & Wagner, E. K. (1994). Molecular analysis of herpes simplex virus type 1 during epinephrine-induced reactivation of latently infected rabbits in vivo. Journal of Virology 68, 1283-1292.[Abstract]
Cook, S. D., Paveloff, M. J., Doucet, J. J., Cottingham, A. J., Sedarati, F. & Hill, J. M. (1991). Ocular herpes simplex virus reactivation in mice latently infected with latency-associated transcript mutants. Investigative Ophthalmology & Visual Science 32, 1558-1561.[Abstract]
Deshmane, S. L., Nicosia, M., Valyi-Nagy, T., Feldman, L. T., Dillner, A. & Fraser, N. W. (1993). An HSV-1 mutant lacking the LAT TATA element reactivates normally in explant cocultivation. Virology 196, 868-872.[Medline]
Devi-Rao, G. B., Bloom, D. C., Stevens, J. G. & Wagner, E. K. (1994). Herpes simplex virus type 1 DNA replication and gene expression during explant-induced reactivation of latently infected murine sensory ganglia. Journal of Virology 68, 1271-1282.[Abstract]
Hill, J. M., Maggioncalda, J. B., Garza, H. H.Jr, Su, Y. H., Fraser, N. W. & Block, T. M. (1996). 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. Journal of Virology 70, 7270-7274.[Abstract]
Hill, J. M., Garza, H. H.Jr, Su, Y. H., Meegalla, R., Hanna, L. A., Loutsch, J. M., Thompson, H. W., Varnell, E. D., Bloom, D. C. & Block, T. M. (1997). A 437-base-pair deletion at the beginning of the latency-associated transcript promoter significantly reduced adrenergically induced herpes simplex virus type 1 ocular reactivation in latently infected rabbits. Journal of Virology 71, 6555-6559.[Abstract]
Ho, D. Y. & Mocarski, E. S. (1989). Herpes simplex virus latent RNA (LAT) is not required for latent infection in the mouse. Proceedings of the National Academy of Sciences, USA 86, 7596-7600.[Abstract]
Izumi, K. M., McKelvey, A. M., Devi-Rao, G., Wagner, E. K. & Stevens, J. G. (1989). Molecular and biological characterization of a type 1 herpes simplex virus (HSV-1) specifically deleted for expression of the latency-associated transcript (LAT). Microbial Pathogenesis 7, 121-134.[Medline]
Javier, R. T., Thompson, R. L. & Stevens, J. G. (1987). Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence. Journal of Virology 61, 1978-1984.[Medline]
Javier, R. T., Stevens, J. G., Dissette, V. B. & Wagner, E. K. (1988). A herpes simplex virus transcript abundant in latently infected neurons is dispensable for establishment of the latent state. Virology 166, 254-257.[Medline]
Junejo, F. & Brown, S. M. (1995). Latent phenotype analysis of three deletion variants of herpes simplex virus type 1 (HSV-1) in mouse model. Journal of Infectious Diseases 171, 1031-1034.[Medline]
Leib, D. A., Bogard, C. L., Kosz-Vnenchak, M., Hicks, K. A., Coen, D. M., Knipe, D. M. & Schaffer, P. A. (1989). A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. Journal of Virology 63, 2893-2900.[Medline]
Maggioncalda, J., Mehta, A., Fraser, N. W. & Block, T. M. (1994). 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. Journal of Virology 68, 7816-7824.[Abstract]
Maggioncalda, J., Mehta, A., Bagasra, O., Fraser, N. W. & Block, T. M. (1996). 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. Journal of Neurovirology 2, 268-278.[Medline]
Natarajan, R., Deshmane, S., Valyi-Nagy, T., Everett, R. & Fraser, N. W. (1991). A herpes simplex virus type 1 mutant lacking the ICP0 introns reactivates with normal efficiency. Journal of Virology 65, 5569-5573.[Medline]
Nesburn, A. B. (1983). Report of the Corneal Disease Panel: Vision Research: A National Plan 19831987. St Louis: C.V. Mosby Co.
Perng, G. C., Dunkel, E. C., Geary, P. A., Slanina, S. M., Ghiasi, H., Kaiwar, R., Nesburn, A. B. & Wechsler, S. L. (1994). The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency. Journal of Virology 68, 8045-8055.[Abstract]
Perng, G. C., Ghiasi, H., Slanina, S. M., Nesburn, A. B. & Wechsler, S. L. (1996). The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1·5 kilobases of the 8·3-kilobase primary transcript. Journal of Virology 70, 976-984.[Abstract]
Perng, G., Jones, C., Ciacci-Zanella, H., Henderson, G., Yukht, A., Slanina, S., Hofman, F., Ghiasi, H., Nesburn, A. & Wechsler, S. (2000a). Virus induced neuronal apoptosis blocked by the herpes simplex virus latency associated transcript (LAT). Science 287, 1500-1503.
Perng, G. C., Slanina, S. M., Yukht, A., Ghiasi, H., Nesburn, A. B. & Wechsler, S. L. (2000b). The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits. Journal of Virology 74, 1885-1891.
Sawtell, N. M. & Thompson, R. L. (1992). Herpes simplex virus type 1 latency-associated transcription unit promotes anatomical site-dependent establishment and reactivation from latency. Journal of Virology 66, 2157-2169.[Abstract]
Steiner, I., Spivack, J. G., Lirette, R. P., Brown, S. M., MacLean, A. R., Subak-Sharpe, J. H. & Fraser, N. W. (1989). Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection. EMBO Journal 8, 505-511.[Abstract]
Thompson, R. L. & Sawtell, N. M. (1997). The herpes simplex virus type 1 latency-associated transcript gene regulates the establishment of latency. Journal of Virology 71, 5432-5440.[Abstract]
Trousdale, M. D., Steiner, I., Spivack, J. G., Deshmane, S. L., Brown, S. M., MacLean, A. R., Subak-Sharpe, J. H. & Fraser, N. W. (1991). In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model. Journal of Virology 65, 6989-6993.[Medline]
Wroblewska, Z., Spivack, J. G., Otte, J., Steiner, I., Brown, M., MacLean, A. & Fraser, N. W. (1991). The HSV-1 latency associated transcript (LAT) variants 1704 and 1705 are glycoprotein C negative. Virus Research 20, 193-200.[Medline]
Received 14 November 2000;
accepted 11 January 2001.