Point mutations in herpes simplex virus type 1 oriL, but not in oriS, reduce pathogenesis during acute infection of mice and impair reactivation from latency

Abstract

In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-I(LR) and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-I(LR) in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-I(LR) exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-I(LR) reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-I(LR)-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

FIG. 1.

Location and sequence alignment of HSV-1 oriL and oriS. (A) Diagram of the HSV-1 genome showing the U_L region flanked by inverted repeats ab and b′a′ and the U_S region flanked by inverted repeats a′c′ and ca. The locations of oriL and both copies of oriS are indicated as open ovals. Two essential E genes, UL29 and UL30, are transcribed divergently from oriL and encode the single-stranded DNA binding protein (ICP8) and DNA Pol, respectively; these transcripts are shown as black arrows beneath the genome. Three IE genes encoding viral regulatory proteins ICP4, -22, and -47 are transcribed divergently from oriS; these transcripts are shown as black arrows beneath the genome. (B) Nucleotide sequences of oriL and oriS. For simplicity and to illustrate sequence homology, the sequence of each origin is shown as a single-stranded palindrome. Black dots indicate single nucleotide differences between oriL and oriS. The designations I, II, and III indicate the locations of OBP binding sites. The two black boxes near the apexes of oriL and oriS indicate functional and degenerate GRE half-sites, respectively (9). The arrows and boxes at the bases of the two palindromes represent the two E genes (encoding ICP8 and DNA Pol) that flank oriL and the three IE genes (encoding ICP4 and ICP22/47) that flank oriS. The distances in nucleotides between the last nucleotide shown and the start of transcription of each origin-flanking gene are shown in parentheses.

FIG. 2.

Tear film titers of wild-type, origin mutant, and rescuant viruses during acute infection of mice. Twenty-six mice per group of 30-g (5- to 6-week-old) male ICR mice were infected in both eyes with 2 × 10⁵ PFU/eye of the indicated virus following corneal scarification. On the indicated days, eyes were swabbed as described in Materials and Methods. Viral titers were determined by standard plaque assay on Vero cell monolayers. The results shown are the means ± standard errors of the means (error bars). Although this experiment was performed comparing all five viruses simultaneously, the results are separated into two graphs for ease of interpretation. (A) Comparison of DoriL-I_LR and DoriL-I_LR-R to wild type. (B) Comparison of DoriS-I and DoriS-I-R to wild type. (Note that wild type is the same in panels A and B.)

FIG. 3.

Titers of wild-type, origin mutant, and rescuant viruses from trigeminal ganglia during acute infection of mice. Fourteen mice per group of 30-g (5- to 6-week-old) male ICR mice were infected in both eyes with 2 × 10⁵ PFU/eye of the indicated virus following corneal scarification. TG were removed and processed on the indicated days as described in Materials and Methods. Viral titers were determined by standard plaque assay on Vero cell monolayers. The results shown are the means ± standard errors of the means (error bars). Although this experiment was performed comparing all five viruses simultaneously, the results are separated into two graphs for ease of interpretation. (A) Comparison of DoriL-I_LR and DoriL-I_LR-R to wild type. (B) Comparison of DoriS-I and DoriS-I-R to wild type. (Note that wild type is the same in panels A and B.)

FIG. 4.

Titers of wild-type, origin mutants, and rescuant viruses from hindbrain during acute infection of mice. Seven mice per group of 30-g (5- to 6-week-old) male ICR mice were infected in both eyes with 2 × 10⁵ PFU/eye of the indicated virus following corneal scarification. Hindbrains were removed and processed on the indicated days as described in Materials and Methods. Viral titers were determined by standard plaque assay on Vero cell monolayers. The results shown are the means ± standard errors of the means (error bars). Although this experiment was performed comparing all five viruses simultaneously, the results are separated into two graphs for ease of interpretation. (A) Comparison of DoriL-I_LR and DoriL-I_LR-R to wild type. (B) Comparison of DoriS-I and DoriS-I-R to wild type. (Note that wild type is the same in panels A and B.)

FIG. 5.

Viral-genome loads in latently infected TG. The number of viral genomes in individual TG of mice latently infected with the indicated virus was determined 30 to 34 days p.i. by competitive PCR. The numbers (n) of TG analyzed are shown below the graph. The results shown are the means ± standard errors of the means (error bars). (Inset) Representative standard curve comparing the relationship between numbers of viral genomes and ratio of HSV RR to competitor. Linear regression was used to evaluate the reliability of the standard curve.

FIG. 6.

Reactivation efficiencies of origin mutants in explant and TG cell cultures. (A) Efficiencies of reactivation from explant cultures of TG latently infected with wild type, DoriL-I_LR, DoriL-I_LR-R, DoriS-I, or DoriS-I-R. TG from mice infected 30 to 34 days p.i. were cut into six to eight pieces and cocultured on Vero cell monolayers. Culture medium was assayed daily for the presence of infectious virus as indicated by cytopathic effects in indicator plates of Vero cells. (B) Efficiencies of reactivation from TG cell cultures. TG cell cultures were prepared by pooling 12 to 16 TG per virus group, gently teasing them apart, treating with collagenase to generate single-cell suspensions, and distributing the resulting cell suspensions into 24-well plates. Culture medium was assayed daily for the presence of infectious virus as indicated by cytopathic effects in indicator plates of Vero cells. Each point in panels A and B represents the cumulative percentage of reactivating TG and TG cell cultures from two to four experiments. The number (n) of TG (A) and the numbers of TG cell culture wells (B) are indicated.

FIG. 7.

Genotypes of reactivated viruses. Total DNA was from Vero cell cultures infected with supernatant fluids from individual wells containing reactivated virus from TG of mice infected 30 to 34 days previously with the wild type, DoriL-I_LR, or DoriS-I. Fragments from restriction digestion of total cellular DNA were separated by agarose gel electrophoresis and transferred to a nylon membrane. Southern blotting was performed using a probe specific for either oriL (A) or for oriS (B). (A) Diagram of oriL and the expected fragment sizes after digestion with BstBI. The horizontal line represents the central portion of the U_L segment of the genome. The vertical black line indicates the location of oriL. Black arrows above the genome represent genes transcribed divergently from oriL. Numbers above the ends of the line indicate map locations in nucleotides. Locations of BstBI sites relative to oriL are indicated. Numbers below enzymes indicate nucleotide positions in the viral genome. Numbers beneath fragments indicate their sizes in kb. Letters in parentheses correspond to the fragments shown in the Southern blot to the right of the diagram. (Note that the 42-bp BstBI fragment “d,” containing oriL apical sequences, was not resolved in these tests). (B) Diagram of both copies of oriS and the expected fragment sizes after digestion with the indicated enzymes. Boxes indicate portions of the internal (IR_S) and terminal repeats (TR_S) flanking the U_S region of the genome. Numbers above boxes indicate map locations in nucleotides. Vertical black lines indicate the locations of oriS. Locations of BstEII and BstBI sites relative to oriS are indicated. Numbers below enzymes indicate nucleotide positions in the viral genome. Numbers beneath fragments indicate their sizes in kb. Letters in parentheses correspond to the fragments shown in the Southern blot to the right of the diagram.