Department of Ophthalmology, School of Medicine, University of California Irvine, Medical Center, Building 55, Room 204, Orange, CA 92868, USA1
Author for correspondence: Guey-Chuen Perng. Fax +1 714 456 5073. e-mail gperng{at}uci.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The HSV-1 genome encodes more than 80 genes. However, the only gene abundantly transcribed during neuronal latency is the latency-associated transcript (LAT) (Rock et al., 1987 ; Stevens et al., 1987
). The primary LAT is 8·3 kb long (Dobson et al., 1989
; Zwaagstra et al., 1990
) and overlaps the ICP0 gene in an antisense direction (Rock et al., 1987
; Stevens et al., 1987
). The primary LAT is highly unstable and difficult to detect, but a highly stable 2 kb LAT, an intron derived from the 8·3 kb LAT (Farrell et al., 1991
), is present in readily detectable amounts during neuronal latency. The LAT also overlaps the ICP34.5 gene in an antisense direction. The LAT and ICP34.5 gene are located in the viral long repeats and are therefore diploid genes.
Differences in the virulence of HSV-1 have been observed between different strains (isolates) (Hill et al., 1987 ). Following ocular infection of rabbits with 2x105 p.f.u. per eye of the McKrae strain of HSV-1, approximately 50% of the animals die (Perng et al., 1994
, 1995
, 1999
). In contrast, ocular infection with the KOS(M) strain of HSV-1 does not result in the death of any rabbits (Hill et al., 1987
; S. L. Wechsler, unpublished results). McKrae can therefore be considered a prototypic virulent HSV-1 strain, while KOS can be considered a prototypic avirulent strain.
Several viral genes have been linked to neurovirulence in infected animals. These genes include ICP34.5 (Perng et al., 1995 ; Thompson & Wagner, 1988
), thymidine kinase (Gordon et al., 1984
), ribonucleotide reductase (Cameron et al., 1988
) and the US3 protein kinase (Kurachi et al., 1993
). ICP34.5 has a major impact on neurovirulence, with some ICP34.5 gene mutants reported to reduce neurovirulence by a factor of 100000 or more (Perng et al., 1995
; Thompson et al., 1983
). ICP34.5 is a low-abundance protein that is essential for efficient replication of the virus in neurons in vivo. Unfortunately, there is no effective antibody currently available for studying ICP34.5. Detection and analysis of the ICP34.5 mRNA has also proven extremely difficult. Thus, our knowledge of ICP34.5 function comes almost exclusively from viral mutants. The requirement for ICP34.5 in HSV-1 replication is cell type- and cell state-dependent (Chou & Roizman, 1992
; Chou et al., 1994
; Perng et al., 1995
). We have shown that spontaneous reactivation of d34.5, a McKrae strain ICP34.5 null mutant, is dose-dependent (Perng et al., 1996b
). No spontaneous reactivation could be detected following ocular infection with the standard dose of 2x105 p.f.u. per eye. However, wild-type spontaneous reactivation levels were seen following infection with 1000-fold more virus. As with other ICP34.5 mutants, d34.5 dramatically reduced neurovirulence and did not result in the death of any rabbits, even at the 1000-fold higher infectious dose. Interestingly, since spontaneous reactivation was at wild-type levels at this infectious dose, these results showed that the phenotypes of spontaneous reactivation and neurovirulence are separable.
To determine whether the reduced virulence phenotype of KOS compared with McKrae might be partially or completely due to differences in the ICP34.5 genes of these two HSV-1 strains, in this report we constructed and studied two chimeric viruses. The first virus, designated 34.5KA, contained one copy of the KOS ICP34.5 gene inserted into an ectopic location in the viral unique long region (between UL37 and UL38) of the McKrae-based mutant d34.5, which has both copies of the ICP34.5 gene deleted (Perng et al., 1995 ). The second virus, designated d34.5KR, contained the ICP34.5 gene of KOS in place of both copies of the McKrae ICP34.5 gene on an otherwise wild-type McKrae genomic background. We also sequenced the ICP34.5 genes of McKrae and KOS to look for potential differences.
We report here that, compared with the McKrae ICP34.5 gene, the KOS ICP34.5 gene supported virus virulence very poorly. In contrast, the KOS ICP34.5 gene supported high levels of spontaneous reactivation of the McKrae strain, even though the KOS strain of HSV-1 does not reactivate in the rabbit model. Thus, the reduced neurovirulence of KOS compared with McKrae appeared to be at least partially due to one or more of the sequence differences that we found in the KOS ICP34.5 gene compared with the McKrae ICP34.5 gene, while the reduced reactivation phenotype of KOS compared with McKrae could not be accounted for by differences in their ICP34.5 genes.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of the KOS ICP34.5 chimeric viruses.
The parental virus for construction of 34.5KA and d34.5KR was d34.5, a McKrae-derived mutant in which both copies of the ICP34.5 gene (one in each long repeat) are deleted. The construction of 34.5KA, containing a single copy of the KOS ICP34.5 gene (a DraISphI restriction fragment corresponding to HSV-1 genomic nt 125990124480) in an ectopic location between the UL37 and UL38 genes was identical to that of 34.5A (Perng et al., 1996a ), except that the ICP34.5 gene fragment was from KOS instead of McKrae. Construction of d34.5KR was identical to that of the marker-rescued virus d34.5R (Perng et al., 1995
), except that the ICP34.5 gene fragment (a Sau3ASphI restriction fragment corresponding to HSV-1 genomic nt 126770124480) used to restore both deleted copies of the ICP34.5 gene was from KOS instead of McKrae. This fragment contains 582 nucleotides of the KOS sequence downstream of the 3' end of the d34.5 deletion and 791 nucleotides of the KOS sequence upstream of the 5' end of the d34.5 deletion. This resulted in sufficient overlapping DNA at both ends of the deletion for homologous recombination to occur. All mutant viruses were triple plaque-purified and their structures were confirmed by extensive restriction enzyme digestion and Southern blot analysis.
Replication in tissue culture.
CV-1 cell monolayers at 7080% confluency were infected with virus at 0·01 p.f.u. per cell and all monolayers were refed with exactly the same amount of MEM containing 10% FCS. Virus was harvested at various times by two cycles of freeze-thawing of the monolayers and medium (from -80 °C to room temperature). The amount of virus in each sample was then determined by standard plaque assay on RS cells.
Animals.
Rabbits were 8- to 10-week-old male New Zealand Whites from Irish Farms. Rabbits were treated in accordance with the Association for Research in Vision and Ophthalmology, the American Association for Laboratory Animal Care and the National Institutes of Health guidelines.
Rabbit model of ocular HSV-1 infection, latency and spontaneous reactivation.
As previously described (Perng et al., 1994 , 1995
), rabbits were bilaterally infected by placing 2x105 p.f.u. of virus per eye into the conjunctival cul-de-sac, closing the eye and rubbing the lid gently against the eye for 30 s. To examine acute replication of virus in rabbit eyes, tear films were collected from five eyes per group, each eye from a different rabbit, on days 3, 5, 7 and 10 post-infection (p.i.). The amount of virus was determined by standard plaque assay. Virulence (or neurovirulence) was defined as death due to viral encephalitis during the first 21 days p.i. Beginning on day 31 p.i., tear films were collected daily from each eye for 26 days using a nylon-tipped swab. The swab was then placed in 0·5 ml of tissue culture medium and squeezed, and the inoculated medium was used to infect RS cell monolayers. These monolayers were observed in a masked fashion by phase light microscopy for up to 5 days for HSV-1 cytopathic effects (CPE). All positive monolayers were blind-passaged on to fresh cells to confirm the presence of virus. DNA was purified from positive cultures and analysed by restriction enzyme digestion and Southern analysis to confirm that the CPE was due to reactivated HSV-1 and that the reactivated virus was identical to the input virus (data not shown).
DNA sequencing.
Purified HSV-1 DNA was used as a template for PCR. Due to the high GC content of the sequence, PCR was performed using a series of primers (MWG Biotech Inc.), which generated 15 overlapping products encompassing the entire ICP34.5 gene. A Clontech Advantage-GC2 polymerase Mix kit (Clontech) was used according to the manufactures protocol. The resultant PCR products were sequenced using the SequiTherm EXCEL II DNA Sequencing Kit (Epicenter Technologies).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Survival of rabbits ocularly infected with 34.5KA and d34.5KR
We have previously reported that d34.5 does not result in the death of any rabbits following ocular infection, even at doses as high as 1x108 p.f.u. per eye (Perng et al., 1996b ). To determine whether virulence (as defined by death following ocular infection) was restored in 34.5KA or d34.5KR, rabbits were ocularly infected with 2x105 p.f.u. of virus per eye, as described above. As expected, all (10/10) of the rabbits infected with d34.5 survived (Fig. 4B
). Also as expected, only about 60% of rabbits infected with wild-type McKrae survived in these two experiments. We have previously shown that 34.5A, containing one copy of the McKrae ICP34.5 gene in an ectopic location, only partially restored virulence. The results shown here (78% survival; Fig. 4A
) are consistent with this. In contrast, 34.5KA did not result in the death of any rabbits (Fig. 4A
). However, because of the intermediate phenotype of 34.5A, it was not clear from the 34.5KA results whether the KOS ICP34.5 gene can substitute for the McKrae ICP34.5 gene as regards the McKrae virulence phenotype.
|
Spontaneous reactivation
We have previously shown that the ICP34.5 deletion mutant d34.5 has significantly reduced spontaneous reactivation following ocular infection of rabbits with 2x105 p.f.u. of virus per eye (Perng et al., 1995 ). We have also shown that 34.5A and d34.5R both have a wild-type McKrae spontaneous reactivation phenotype (Perng et al., 1995
, 1996a
). To examine spontaneous reactivation of 34.5KA and d34.5KR (the corresponding chimeric viruses containing the KOS ICP34.5 gene in place of the McKrae ICP34.5 gene), all eyes from the surviving rabbits in the experiments shown in Fig. 4(A)
and (B)
were swabbed daily to collect tear films for analysis of spontaneously reactivated virus as described in Methods. Eye-swab collection began 31 days p.i., at which time latency had already been established (Perng et al., 1994
). The cumulative number of virus-positive tear film cultures (indicative of spontaneously reactivated virus) during the 26 day study period are shown in Fig. 5
. As expected, d34.5 appeared to have a dramatically reduced spontaneous reactivation rate compared with wild-type McKrae (Fig. 5B
), consistent with our previous findings. In contrast, 34.5KA and 34.5A (Fig. 5A
) and d34.5KR (Fig. 5B
) all appeared to have a wild-type McKrae spontaneous reactivation phenotype.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations that delete or significantly alter the ICP34.5 gene greatly reduce virus virulence as measured by both peripheral and intracranial challenge (Perng et al., 1995 ; Thompson et al., 1983
). Reactivation is also greatly reduced in these mutants following infection with the doses of virus typically used (i.e. approximately 2x105 to 2x106 p.f.u.) (Perng et al., 1995
). To determine whether a defect(s)' in the KOS ICP34.5 gene might account for either the inability of KOS to reactivate spontaneously in the rabbit eye model or for avirulence of KOS in this model, we constructed and reported here on two different chimeric viruses. Both chimeric viruses were constructed on the genomic background of the McKrae ICP34.5 null mutant d34.5. Both copies of the ICP34.5 gene (one in each long repeat) are deleted and, like KOS, d34.5 is avirulent and has a negative spontaneous reactivation phenotype following infection of rabbits with 2x105 p.f.u. of virus per eye. To construct the first chimeric virus, 34.5KA, one copy of the KOS ICP34.5 gene was inserted into an ectopic location in the unique long region of d34.5. This chimeric virus was identical to our previous mutant 34.5A, except that in 34.5A the ectopic ICP34.5 gene was from McKrae rather than KOS. The second chimeric virus, d34.5KR, was constructed by marker rescue of both ICP34.5 gene deletions in d34.5 using a small (2·3 kb) piece of KOS DNA. d34.5KR was identical to d34.5R (marker-rescued d34.5 using McKrae DNA) and also to wild-type McKrae, except that the ICP34.5 gene (and an indeterminate amount of flanking DNA, but less than 1 kb on each side of the gene) was from KOS.
In the experiments presented here, the KOS ICP34.5 gene successfully replaced the McKrae ICP34.5 gene and produced McKrae-like phenotypes for replication in CV-1 cells, replication in rabbit eyes and spontaneous reactivation. These results demonstrate that the KOS ICP34.5 gene is capable of supporting the high-level spontaneous reactivation phenotype when placed in an appropriate virus genetic background. Thus, defects' in ICP34.5 cannot account for the negative spontaneous reactivation phenotype of KOS in rabbits. Differences in LAT also cannot account for the reduced spontaneous reactivation phenotype of KOS, since we have previously shown that a chimeric virus in which the functional portion of the McKrae LAT gene was replaced with the corresponding portion of the KOS LAT had a McKrae spontaneous reactivation phenotype (Drolet et al., 1998 ). Thus, both the KOS LAT and the KOS ICP34.5 genes are capable of supporting a McKrae-like spontaneous reactivation phenotype even though KOS itself does not reactivate spontaneously.
In contrast to the findings for replication in CV-1 cells and rabbit eyes and the spontaneous reactivation phenotype, the KOS ICP34.5 gene was not able to substitute efficiently for the McKrae ICP34.5 gene for the phenotype of virus virulence. This strongly suggests that differences between the KOS and McKrae ICP34.5 genes may play an important role in the low virus virulence of KOS compared with McKrae. However, they do not rule out the possibility that differences in additional KOS genes may also be significant. In fact, there are at least three reports indicating that a defect in gB significantly contributes to the reduced pathogenicity of KOS (Kosovsky et al., 2000 ; Kostal et al., 1994
; Yuhasz & Stevens, 1993
). These results also confirm our previous findings that the phenotype of virus virulence can be separated from both the spontaneous reactivation phenotype and the ability of the virus to replicate efficiently in rabbit eyes (Perng et al., 1996a
, b
).
To look for differences in the KOS ICP34.5 gene that might alter its ability to support the virus virulence phenotype, we sequenced the ICP34.5 gene from KOS and McKrae and compared these sequences to each other and to 17syn+. Since 17syn+ and McKrae have similar virus virulence phenotypes, it seems logical to assume that if the KOS sequence differs from both McKrae and 17syn+, this difference may affect the virus virulence phenotype. Several differences were noted between the KOS and McKrae sequences for the ICP34.5 protein. The predicted ICP34.5 protein sequence contains an N-terminal arginine (R)-rich cluster and a more central prolinealaninethreonine (PAT) repeat region. It has been proposed that one or both of these regions is involved in cellular localization of the ICP34.5 protein and in its neurovirulence properties (Mao & Rosenthal, 2002 ). The arginine cluster of the McKrae, KOS and 17syn+ ICP34.5 proteins are similar, containing eight, seven and nine arginines, respectively. This small range of differences is not thought to be significant (Mao & Rosenthal, 2002
). In contrast, McKrae contains nine PAT repeats while KOS contains only five PAT repeats, suggesting that the reduced number of PAT repeats in KOS may account for the decreased virulence properties of its ICP34.5 gene. However 17syn+, which has virulence and reactivation properties similar to those of McKrae, has six PAT repeats, only one more than KOS.
Compared with McKrae and 17syn+, the KOS ICP34.5 amino acid sequence differs in two additional locations. At the location corresponding to amino acid 188 of McKrae, KOS contains a threonine, while McKrae and 17syn+ both contain an alanine. Compared with McKrae and 17syn+, KOS has a C nucleotide inserted into the DNA sequence after the nucleotide corresponding to McKrae nt 692 and a C deleted from the DNA sequence at McKrae nt 754. This produces a frame shift at the location corresponding to aa 233251 of McKrae. This results in a stretch of 19 out of 20 mismatched amino acids in the KOS sequence compared with both McKrae and 17syn+. At this time it is unknown which of the difference(s) in the KOS sequence results in the inability of the KOS ICP34.5 protein to support the high virus virulence phenotype.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chou, J. & Roizman, B. (1992). The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programmed cell death in neuronal cells. Proceedings of the National Academy of Sciences, USA 89, 3266-3270.[Abstract]
Chou, J., Poon, A. P., Johnson, J. & Roizman, B. (1994). Differential response of human cells to deletions and stop codons in the gamma(1)34.5 gene of herpes simplex virus. Journal of Virology 68, 8304-8311.[Abstract]
Dobson, A. T., Sederati, F., Devi-Rao, G., Flanagan, W. M., Farrell, M. J., Stevens, J. G., Wagner, E. K. & Feldman, L. T. (1989). Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus. Journal of Virology 63, 3844-3851.[Medline]
Drolet, B. S., Perng, G. C., Cohen, J., Slanina, S. M., Yukht, A., Nesburn, A. B. & Wechsler, S. L. (1998). The region of the herpes simplex virus type 1 LAT gene involved in spontaneous reactivation does not encode a functional protein. Virology 242, 221-232.[Medline]
Farrell, M. J., Dobson, A. T. & Feldman, L. T. (1991). Herpes simplex virus latency-associated transcript is a stable intron. Proceedings of the National Academy of Sciences, USA 88, 790-794.[Abstract]
Gordon, Y. J., Simon, P. L. & Armstrong, J. A. (1984). Neurovirulence of an herpes simplex type 1 thymidine kinase negative mutant determined by virus biochemical defect and host immune system in mice. Brief report. Archives of Virology 80, 225-229.[Medline]
Hill, J. M., Rayfield, M. A. & Haruta, Y. (1987). Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits. Current Eye Research 6, 91-97.[Medline]
Kosovsky, J., Vojvodova, A., Oravcova, I., Kudelova, M., Matis, J. & Rajcani, J. (2000). Herpes simplex virus 1 (HSV-1) strain HSZP glycoprotein B gene: comparison of mutations among strains differing in virulence. Virus Genes 20, 27-33.[Medline]
Kostal, M., Bacik, I., Rajcani, J. & Kaerner, H. C. (1994). Replacement of glycoprotein B gene in the herpes simplex virus type 1 strain ANGpath DNA by that originating from nonpathogenic strain KOS reduces the pathogenicity of recombinant virus. Acta Virologica 38, 77-88.[Medline]
Kurachi, R., Daikoku, T., Tsurumi, T., Maeno, K., Nishiyama, Y. & Kurata, T. (1993). The pathogenicity of a US3 protein kinase-deficient mutant of herpes simplex virus type 2 in mice. Archives of Virology 133, 259-273.[Medline]
McGeoch, D. J. (1987). The genome of herpes simplex virus: structure, replication and evolution. Journal of Cell Science Supplement 7, 67-94.[Medline]
Mao, H. & Rosenthal, K. S. (2002). An N-terminal arginine-rich cluster and a prolinealaninethreonine repeat region determine the cellular localization of the herpes simplex virus type 1 ICP34.5 protein and its ligand, protein phosphatase 1. Journal of Biological Chemistry 277, 11423-11431.
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., Thompson, R. L., Sawtell, N. M., Taylor, W. E., Slanina, S. M., Ghiasi, H., Kaiwar, R., Nesburn, A. B. & Wechsler, S. L. (1995). An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation. Journal of Virology 69, 3033-3041.[Abstract]
Perng, G. C., Chokephaibulkit, K., Thompson, R. L., Sawtell, N. M., Slanina, S. M., Ghiasi, H., Nesburn, A. B. & Wechsler, S. L. (1996a). The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation. Journal of Virology 70, 282-291.[Abstract]
Perng, G. C., Ghiasi, H., Slanina, S. M., Nesburn, A. B. & Wechsler, S. L. (1996b). High-dose ocular infection with a herpes simplex virus type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation. Journal of Virology 70, 2883-2893.[Abstract]
Perng, G. C., Ghiasi, H., Slanina, S. M., Nesburn, A. B. & Wechsler, S. L. (1996c). 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. C., Slanina, S. M., Yuhkt, A., Drolet, B. S., Keleher, W. J., Ghiasi, H., Nesburn, A. B. & Wechsler, S. L. (1999). A herpes simplex virus type 1 latency associated transcript (LAT) mutant with increased virulence and reduced spontaneous reactivation. Journal of Virology 73, 920-929.
Perry, L. J. & McGeoch, D. J. (1988). The DNA sequences of the long repeat region and adjoining parts of the long unique region in the genome of herpes simplex virus type 1. Journal of General Virology 69, 2831-2846.[Abstract]
Rock, D. L., Nesburn, A. B., Ghiasi, H., Ong, J., Lewis, T. L., Lokensgard, J. R. & Wechsler, S. L. (1987). Detection of latency-related viral RNAs in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1. Journal of Virology 61, 3820-3826.[Medline]
Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. & Feldman, L. T. (1987). RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons. Science 235, 1056-1059.[Medline]
Thompson, R. L. & Wagner, E. K. (1988). Partial rescue of herpes simplex virus neurovirulence with a 3·2 kb cloned DNA fragment. Virus Genes 1, 261-273.[Medline]
Thompson, R. L., Wagner, E. K. & Stevens, J. G. (1983). Physical location of a herpes simplex virus type-1 gene function(s) specifically associated with a 10 million-fold increase in HSV neurovirulence. Virology 131, 180-192.[Medline]
Yuhasz, S. A. & Stevens, J. G. (1993). Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness. Journal of Virology 67, 5948-5954.[Abstract]
Zwaagstra, J. C., Ghiasi, H., Slanina, S. M., Nesburn, A. B., Wheatley, S. C., Lillycrop, K., Wood, J., Latchman, D. S., Patel, K. & Wechsler, S. L. (1990). Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript. Journal of Virology 64, 5019-5028.[Medline]
Received 27 May 2002;
accepted 19 July 2002.