A recombinant herpes simplex virus type 1 expressing two additional copies of gK is more pathogenic than wild-type virus in two different strains of mice

Abstract

The effect of glycoprotein K (gK) overexpression on herpes simplex virus type 1 (HSV-1) infection in two different strains of mice was evaluated using a recombinant HSV-1 virus that expresses two additional copies of the gK gene in place of the latency-associated transcript (LAT). This mutant virus (HSV-gK3) expressed higher levels of gK than either the wild-type McKrae virus or the parental dLAT2903 virus both in vitro (in cultured cells) and in vivo (in infected mouse corneas and trigeminal ganglia [TG] of BALB/c and C57BL/6 mice). gK transcripts were detected in the TG of both HSV-gK3-infected mouse strains on day 30 postinfection (p.i.), while gB transcripts were detected only in the TG of the HSV-gK3-infected C57BL/6 mice, a finding that suggests that increased gK levels promote chronic infection. C57BL/6 mice infected with HSV-gK3 also contained free virus in their TG on day 30 p.i. Both HSV-gK3-infected mouse strains had significantly higher corneal scarring (CS) than did McKrae-infected mice. T-cell depletion studies in C57BL/6 mice suggested that this CS enhancement in the HSV-gK3-infected mice was mediated by a CD8+ T-cell response. Taken together, these results strongly suggest that increased gK levels promote eye disease and chronic infection in infected mice.

FIG. 1.

Construction and structure of the HSV-gK³ mutant virus. (A) The top schematic diagram shows the HSV-1 McKrae genome in the prototypic orientation. TR_L and IR_L represent the terminal and internal (or inverted) long repeats, respectively, and TR_S and IR_S represent the terminal and internal (or inverted) short repeats, respectively. U_L and U_S represent the long and short unique regions, respectively. The solid rectangle represents the very stable 2-kb LAT. The start site for LAT transcription is indicated by the arrow at +1. “TATA” designates the relative location of the LAT promoter TATA box 28 nt upstream of the start of transcription. (B) dLAT2903 has a deletion from LAT nt −161 to +1667 in both copies of LAT and makes no LAT RNA. (C) HSV-gK³ was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural gK gene [including its 3′ poly(A) signal] as described in Materials and Methods. (D) HSV-gK³ Southern blot. Subconfluent RS cell monolayers were infected with 10 PFU/cell of HSV-gK³ or McKrae for 16 h. Viral DNAs were isolated, and 5 μg of DNA from each virus was digested with EcoRI/EcoRV and hybridized to ³²P-labeled HSV-1 gK (HSV-1 nt 112170 to 113193). As expected, only one fragment is seen in the McKrae lane. This band also is observed in HSV-gK³ and corresponds to HSV-1 nt 110094 to 118640 surrounding the naturally occurring gK (HSV-1 nt 112170 to 113193). In contrast to the McKrae lane, in the HSV-gK³ lane, in addition to the natural gK, there are two additional bands corresponding to two extra copies of the gK gene inserted within IR_L (HSV-1 nt 110094 to 118640) and TR_L (HSV-1 nt 13 to 7727) regions of dLAT2903. Both regions within the IR_L and TR_L have a deletion of approximately 1,600 bp of LAT.

FIG. 2.

gK and gB transcriptions in infected RS cells. Subconfluent RS cell monolayers in quadruplicate were infected with 10 PFU/cell of HSV-gK³ or dLAT2903 for 8 and 16 h. Total RNA was isolated from these infected RS cells, and TaqMan RT-PCR was performed as described in Materials and Methods using gB- and gK-specific primers. The 18S rRNA expression was used to normalize the relative expression of gK and gB mRNAs in HSV-gK³- and dLAT2903-infected RS cells. PI, postinfection. Each point represents the mean ± standard error of the mean (n = 4).

FIG. 3.

Quantitation of gK and gB mRNAs in vivo. (A) gK and gB transcriptions in TG. BALB/c or C57BL/6 mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³ or McKrae virus. TG from individual mice were isolated 5 days p.i. (PI), and TaqMan RT-PCR was performed as described in Materials and Methods. GAPDH expression was used to normalize the relative expression of gK and gB mRNA in HSV-gK³- and McKrae-infected TGs. Each point represents the mean ± standard error of the mean from five mice. (B) gK and gB transcription in corneas. Individual mouse corneas infected as described above were harvested, and TaqMan RT-PCRs for gK and gB transcripts were performed as described above. Each point represents the mean ± standard error of the mean from 5 mice.

FIG. 4.

Replication of HSV-gK³ in vitro and in vivo. (A) Virus replication in RS cells. Subconfluent RS cell monolayers in triplicate from two separate experiments were infected with 10 PFU/cell of HSV-gK³, dLAT2903, McKrae, or HSV-gK³R as described in Materials and Methods. Total virus was harvested at the indicated times p.i. by two cycles of freeze-thawing. The amount of virus at each time point was determined by standard plaque assays on RS cells. Each point represents the mean ± standard error of the mean (n = 6). (B and C) Virus replication in mouse tears. BALB/c (B) or C57BL/6 (C) mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³, dLAT2903, or McKrae virus. Tear films were collected from days 1 to 10, and virus titers were determined by standard plaque assays. Each point represents the mean titers of 40 eyes from two separate experiments for days 1 to 5 and 20 eyes from two separate experiments for days 6 to 10. (D) Virus titers in TG. BALB/c mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³, dLAT2903, or McKrae virus as described above. Mice were euthanized on the indicated days, TG from each mouse were removed, combined, and homogenized, and virus titers were determined for the TG from each mouse. Each bar represents the mean ± standard error of the mean of the TG from 5, 15, and 5 mice for days 3, 5, and 6 p.i., respectively.

FIG. 5.

Eye diseases in mice infected with HSV-gK³. BALB/c and C57BL/6 mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³ or McKrae virus and were used to determine blepharitis and CS. Blepharitis and CS in surviving mice were examined on days 7 and 30 p.i. as described in Materials and Methods. In the BALB/c mice, the blepharitis and CS scores represents the average ± standard error of the mean from 100 and 22 eyes, respectively, from HSV-gK³-infected mice and 100 and 36 eyes, respectively, from McKrae-infected mice. In C57BL/6 mice, the blepharitis and CS scores represent the average ± standard error of the mean from 78 eyes for HSV-gK³-infected mice and 60 eyes for McKrae-infected mice.

FIG. 6.

Estimation of CD4 and CD8 copy numbers in corneas and TG of infected mice. BALB/c or C57BL/6 mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³ or McKrae virus. Corneas and TG from individual mice were isolated on day 5 or 30 p.i. (PI), and TaqMan RT-PCR was performed on total RNA isolated from those tissues as described in Materials and Methods. GAPDH was used as an endogenous control to normalize the relative expression of CD4 and CD8 mRNA in HSV-gK³- and McKrae-infected corneas and TG. Each point represents the mean ± standard error of the mean from 5 mice. (A) CD4 and CD8 mRNAs in mouse corneas on day 5 p.i. (B) CD4 and CD8 mRNAs in mouse TG on day 5 p.i. (C) CD4 and CD8 mRNAs in mouse corneas on day 30 p.i. (D) CD4 and CD8 mRNAs in mouse TG on day 30 p.i.

FIG. 7.

CS in C57BL/6 mice is CD8⁺ T-cell dependent. Ten C57BL/6 mice were depleted of their CD4⁺ or CD8⁺ T cells prior to ocular infection with HSV-gK³ or McKrae virus as described in Materials and Methods. Mock control mice were depleted with irrelevant monoclonal antibody. CS was measured 30 days after ocular infection in surviving mice. For each bar, CS (y axis) represents the average of the scarring from 16 eyes (CD4⁺ depleted), 18 eyes (CD8⁺ depleted), or 20 eyes (mock depleted). The error bars indicate the standard error of the mean.

FIG. 8.

Detection of gK and gB transcripts in the TG during latency. BALB/c or C57BL/6 mice were ocularly infected with 2 × 10⁵ PFU/eye of HSV-gK³ or McKrae virus. TG from individual mice were isolated 30 days p.i. (PI), and TaqMan RT-PCR was performed as described in Materials and Methods. GAPDH was used as an endogenous control to normalize the relative expression of gK and gB mRNAs in HSV-gK³-and McKrae-infected TG. Each point represents the mean ± standard error of the mean from 5 mice. The numbers in parentheses on top of each bar indicate the number of positive mouse TG per total mouse TG tested for each transcript.