The effect of latency-associated transcript on the herpes simplex virus type 1 latency-reactivation phenotype is mouse strain-dependent

Guey-Chuen Perng1, Susan M. Slanina1, Homayon Ghiasi1,2, Anthony B. Nesburn1,2 and Steven L. Wechsler1,2

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
Top
Abstract
Main text
References
 
Herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) null mutants reactivate poorly in the rabbit ocular model. The situation in mice is less clear. Reports concluding that LAT null mutants reactivate poorly in the mouse explant-induced reactivation (EIR) model are contradicted by a similar number of reports of normal EIR of LAT- mutants in mice. To determine if the EIR phenotype might be mouse strain-dependent we infected BALB/c and Swiss Webster mice with LAT- or LAT+ virus and assessed EIR in individual trigeminal ganglia. Compared to LAT+ virus, LAT- virus reactivated poorly in Swiss Webster mice (P<0·05). In contrast, the EIR phenotype of these viruses was similar in BALB/c mice (P>0·1). Thus, LAT appeared to have a much greater impact on the EIR phenotype in Swiss Webster mice than in BALB/c mice. The mouse strain therefore appeared consequential in the HSV-1 EIR phenotype in mice.


   Main text
Top
Abstract
Main text
References
 
Following ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up nerves and establishes life-long latent infection in nuclei of sensory neurons of the trigeminal ganglia (TG). At various times the virus may reactivate, travel back to the eye, and produce recurrent disease. This can produce scarring of the cornea and loss of vision, making recurrent HSV-1 a major cause of infectious corneal blindness in the developed world (Nesburn, 1983 ). A similar HSV latency–reactivation–recurrent disease cycle produces recurrent ‘cold sores’ in and around the mouth and recurrent genital lesions.

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(A–H). 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).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1. Kinetics of EIR. BALB/c or Swiss Webster mice were infected in both eyes with the indicated doses of LAT- or LAT+ virus (panels A–H). Thirty days post-infection, a time at which latency is already established, mice were euthanized and TG were removed. Individual TG (two per mouse) were incubated in tissue culture media (Eagle’s minimum essential medium with 10% foetal bovine serum) at 37 °C. An aliquot was removed from each culture daily for 18 days and used to infect rabbit skin (RS) cell monolayers. The RS cells were observed for the appearance of CPE for up to 2 weeks to determine the time of first appearance of reactivated viruses from each TG. The results are plotted as the cumulative percentage of TG that had reactivated. The P values were determined from survival curves (log rank test) using GraphPad Prism version 3.00 for Windows (GraphPad Software) The results from panels (A) to (H), along with results using 102 p.f.u./eye, are compiled in panels (I) and (J). Asterisks indicate a significant difference between LAT+ and LAT- as shown in panels (A)–(H). The numbers of TG in each group are shown in parentheses.

 
In contrast, in Swiss Webster mice EIR of the LAT- virus was significantly reduced compared to the LAT+ virus at all infectious doses from 103 to 106 p.f.u./eye (Fig. 1E–H; summarized in Fig. 1J). These results are consistent with those we previously reported for dLAT2903 in the rabbit (Perng et al., 1994 ).

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.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Replication in eyes. Mice were ocularly infected without corneal scarification. Tear films were collected with nylon swabs at various times post-infection and the amount of virus was determined by plaque assay, as we have previously described (Perng et al., 1994 ). Each point represents the average titre from five eyes, each from a different mouse. Panel (A): BALB/c mice infected with 105 p.f.u./eye; (B) Swiss Webster mice infected with 105 p.f.u./eye; (C) BALB/c mice infected with 106 p.f.u./eye; (D) Swiss Webster mice infected with 106 p.f.u./eye. {bullet}, LAT+; {circ}, LAT-.

 
Survival was followed for 21 days post-infection in the mice used for the above EIR studies (Fig. 3). Compared to BALB/c mice, Swiss Webster mice appeared to be more resistant to lethal ocular challenge with LAT+ (Fig. 3D, E, F; P=0·01 and P=0·02, chi-squared, for 105 and 106 p.f.u./eye respectively; P=0·009, paired Student’s t-test for the range 103 to 106 p.f.u./eye) and LAT- virus (Fig. 3G, P=0·02 paired t-test over the range 104 to 106 p.f.u./eye). Although the results in Fig. 3(H, I) show a trend for LAT- virus to be less virulent (i.e. increased survival) than the LAT+ virus, especially at lower infectious doses, the differences were not significant (P>0·05 at all individual infectious doses and by paired t-test). This is consistent with our previous findings that the LAT+ and LAT- viruses have similar virulence in rabbits (Perng et al., 1994 ).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3. Survival of BALB/c and Swiss Webster mice following ocular infection with LAT- or LAT+ virus. Panels (A)–(E), mice were infected in both eyes with the indicated dose of virus and survival was followed for 21 days. The numbers over each bar indicate the no. of surviving mice/no. of mice infected. P values determined by chi-squared, are shown within each infectious dose only for those results that were significantly different (groups being compared are indicated by the lines above the bars). Panels (F)–(I), additional analyses of the results shown in (A)–(E). P values are based on paired Student’s t-tests comparing infectious doses 103–106 in (F); 104–106 in (G); 103–105 in (H); and 103–104 in (I).

 
As discussed above, there is a lack of consistency in the literature regarding the effect of LAT on the HSV-1 EIR phenotype in mice. The results presented here may help to explain this discrepancy. Our findings suggest that in some mouse strains LAT has a major impact on the EIR phenotype, while in other mouse strains LAT has limited or no impact. Thus, differences in the mouse strains used to test LAT mutants may partially account for discrepancies in the literature. In addition, in BALB/c mice infected at 104 or 105 p.f.u./eye there appeared to be a nonsignificant tendency towards decreased EIR with the LAT- virus, while at 106 p.f.u./eye there was no hint of any difference between the LAT- and LAT+ viruses. Thus, if LAT does play a role in the EIR phenotype in BALB/c mice, the effect may be dependent on infectious dose, with 106 p.f.u./eye being too high a dose. Since infectious doses in the range of 105 to 106 p.f.u./eye are typically employed in HSV-1 mouse experiments, even small differences in the infectious doses used in different studies may also partially account for the discrepancies in the literature. In addition, since the difference between LAT- and LAT+ in BALB/c mice approached significance at infectious doses of 104 and 105 p.f.u./eye, it is possible that, by chance, statistical significance might be achieved in some experiments but not in others, especially if small numbers of mice are used. All of these factors may contribute to the lack of consistency in the literature regarding the effect of LAT effect on the EIR phenotype in mice.

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
 
This work was supported by Public Health Service grants EY07566, EY11629 and EY12823, the Discovery Fund for Eye Research, and the Skirball Program in Molecular Ophthalmology. The authors thank Anita Avery for her expert technical support.


   References
Top
Abstract
Main text
References
 
Block, T. M., Spivack, J. G., Steiner, I., Deshmane, S., McIntosh, M. T., Lirette, R. P. & Fraser, N. W. (1990). A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection. Journal of Virology 64, 3417-3426.[Medline]

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 1983–1987. 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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.