A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels - PubMed (original) (raw)

. 2002 Feb;76(3):1224-35.

doi: 10.1128/jvi.76.3.1224-1235.2002.

Barak Maguen, Ling Jin, Kevin R Mott, Nelson Osorio, Susan M Slanina, Ada Yukht, Homayon Ghiasi, Anthony B Nesburn, Melissa Inman, Gail Henderson, Clinton Jones, Steven L Wechsler

Affiliations

A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels

Guey-Chuen Perng et al. J Virol. 2002 Feb.

Abstract

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500-1503, 2000; M. Inman et al., J. Virol. 75:3636-3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (P < 0.0004 and P = 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.

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Figures

FIG. 1.

FIG. 1.

Schematic representation of mutant viruses. (A) Genomic structure of wild-type HSV-1 strain McKrae. TRL and IRL indicate the viral long repeats (terminal and internal, respectively). IRS and TRS indicate the viral short repeats. UL and US indicate the long and short unique regions. The dashed lines indicate that the region of the TRL and IRL are expanded below with the TRL inverted relative to the IRL so that both identical regions can be represented by a single image. (B) The LAT region of wild-type and marker-rescued dLAT2903R. The LAT promoter is represented by the open rectangle. The primary 8.3-kb LAT transcript is indicated by the large arrow. The solid rectangle indicates the stable 2-kb LAT intron. The start of LAT transcription is indicated by the arrow at +1 (LAT nucleotide 1). The relative locations of the ICP0 and ICP34.5 RNAs are shown for reference. (C) The deletion from LAT nucleotide −161 to +1667 in dLAT2903 is indicated by “XXXXXXX.” dLAT2903 is a true LAT null mutant that is missing primary LAT promoter elements between positions −161 and +1. dLAT2903 is also lacking a putative secondary LAT promoter, LAP2, located within the 5′ end of the primary LAT transcript prior to the start of the 2-kb LAT (11). This mutant therefore is not capable of expressing any LAT RNA (21). (D) The LAT region of CJLAT. The complete LR gene of BHV-1, including the promoter (a 1,941-bp _Hin_dIII-_Sal_I fragment), was inserted into the deletion in dLAT2903. The resulting virus expresses the LR-RNA from the HSV-1 LAT region.

FIG. 2.

FIG. 2.

RT-PCR and Northern blot analysis of LR-RNA expressed in CJLAT. (A) CV-1 cells were infected with CJLAT, dLAT2903, or wild-type McKrae at an MOI of 5. Total RNA was isolated at various times p.i., and RT-PCR was performed as described in Materials and Methods. Lanes: 1, McKrae (6 h pi); 2, McKrae (24 h p.i.); 3, dLAT2903 (6 h p.i.); 4, dLAT2903 (24 h p.i.); 5, CJLAT (6 h p.i.); 6, CJLAT (12 h p.i.); 7, CJLAT (24 h p.i.); 8, McKrae (48 h p.i.); 9, dLAT2903 (48 h p.i.); 10, CJLAT (48 h p.i.); 11, mock infected; 12, no RNA; 13, plasmid containing LR gene; M, 100-bp marker ladder (the fastest-migrating band is 100 bp). The expected size of the RT-PCR product is 192 bp. (B) No-RT controls. Lanes: 1 to 10 are the same as in panel A, except that no reverse transcriptase was added to the reactions. (C) Total RNA from CV-1 cells infected as described above was isolated 12 h p.i. and analyzed by Northern blot with a _Hin_dIII-_Sal_I restriction fragment probe corresponding to the entire BHV-1 LR gene as described in Materials and Methods. The arrow points to the LR-RNA band. (D) The membrane from panel C was stripped and reprobed with an HSV-1 _Hpa_I-_Mlu_I DNA restriction fragment (LAT nucleotides 1667 to 2850) capable of hybridizing to both LAT and ICP0 (arrows). CJLAT, CJLAT-infected CV-1 cells; wt, wild-type McKrae-infected CV-1 cells; dLAT2903, dLAT2903-infected CV-1 cells; CV-1, uninfected CV-1 cells.

FIG. 3.

FIG. 3.

Replication of CJLAT in tissue culture. CV-1 cells were infected at an MOI of 0.01. The infected monolayers were harvested by freeze-thawing at the indicated times, and the amounts of infectious virus were determined by plaque assays as described in Materials and Methods.

FIG. 4.

FIG. 4.

Replication of CJLAT in rabbit eyes. Rabbits were infected as described in Materials and Methods. Tears were collected from 10 eyes/group on the days indicated and the amount of infectious virus was determined as described above. Panels A and B are from two independent experiments.

FIG. 5.

FIG. 5.

Rabbit survival. The numbers above each bar indicate the number of surviving rabbits on day 21 p.i. over the number of rabbits initially infected. P values were determined by chi-square analysis. Bars: wt, wild-type McKrae; dLAT, dLAT2903; CJLAT, CJLAT.

FIG. 6.

FIG. 6.

Spontaneous reactivation. Rabbits were infected as described above, and individual sera were collected on day 59 p.i. (A), day 60 p.i. (B), or on the days indicated (C). In panels A and B, each datum point indicates the neutralizing antibody titer of one sera. In panel C, each datum point is the average of between 5 and 10 sera. Panels A and B are from separate experiments. Panels B and C represent sera from the same set of rabbits. wt, wild-type McKrae; dLAT, dLAT2903.

FIG. 7.

FIG. 7.

Replication of CJLAT in mouse eyes and TG. Mice were ocularly infected as described in Materials and Methods. (A) Tears were collected from 10 eyes/group on the indicated days, and the amounts of infectious virus were determined by plaque assay. (B) Mice were infected as in panel A. Five mice/group were euthanatized on the days indicated, and individual TG (10/group/time point) were homogenized, and the amounts of infectious virus were determined by plaque assay.

FIG. 8.

FIG. 8.

Mouse survival. Mice were ocularly infected with 2 × 106 PFU of virus/eye, and survival was determined on day 21 p.i. Panels A and B are from separate experiments. wt, wild-type McKrae; dLATR, dLAT2903R-infected mice.

FIG. 9.

FIG. 9.

Explant reactivation. Ten mice from each of the groups shown in Fig. 8 were selected randomly, and the TG were removed on day 30 p.i. and individually incubated in tissue culture media at 37°C. Aliquots of the tissue culture media were removed daily for up to 21 days and inoculated onto RS cell monolayers to look for the presence of reactivated virus.

References

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