Genome replication and progeny virion production of herpes simplex virus type 1 mutants with temperature-sensitive lesions in the origin-binding protein

Abstract

Genome replication of herpes simplex viruses (HSV) in cultured cells is thought to be started by the action of the virus-encoded origin-binding protein (OBP). In experiments using two HSV-1 mutants with temperature-sensitive lesions in the helicase domain of OBP, we demonstrated that this function is essential during the first 6 hours of the lytic cycle. Once DNA synthesis has started, this function is no longer required, suggesting that origin-driven initiation of viral DNA replication is a single event rather than a continuous process.

FIG. 1.

Kinetics of viral DNA synthesis and influence of temperature upshift. A series of petri dishes (10 cm²) with approximately one million Vero cells each was infected with viruses at an MOI of 10. After incubation at the permissive temperature of 33°C for the time in hours shown on the abscissas, cells together with the culture media were harvested and frozen at −70°C. Total DNA was isolated, aliquots were applied to nitrocellulose membranes, and HSV-specific sequences were quantitated by dot hybridization (21) with a radioactively labeled HSV-1 DNA probe. Signals were calculated as genome equivalents and are symbolized by open circles (○) in the diagrams, which represent the kinetics of the accumulation of viral DNA at the permissive temperature. A parallel series of cell cultures was infected identically, yet after initial incubations at the permissive temperature for the shown periods of time, individual cultures, instead of being harvested immediately, were transferred to the nonpermissive temperature (39°C). The time points of these transfers (upshift) are symbolized by filled triangles (▴). The upshifted cultures were finally harvested altogether at 30 h p.i. Total DNA was isolated and HSV-specific DNA was quantitated as described for the aforementioned samples. The HSV genome equivalents of the shift kinetics experiment are also symbolized by the filled triangles.

FIG. 2.

Kinetics of virion synthesis and influence of temperature upshift. The samples harvested in the experiments described in the legend to Fig. 1 were analyzed for the presence and quantity of infectious particles by plaque titration on Vero cell cultures at 33°C. The open circles (○) represent the kinetics of growth at the permissive temperature, whereas the filled triangles (▴) indicate the yields of infectious particles reached at 30 h postinfection when the infected culture was first kept at 33°C for the shown period of time (▴) and then shifted to 39°C (shift kinetics).

FIG. 3.

Growth of wild-type virus after coinfection with ts mutants. (A) Vero cells were inoculated with tsR, tsS, tsRrev, tsSrev, tsA, tsO, or tsH at an MOI of 5 and simultaneously coinfected with HSV-1 wild type at an MOI of 1.0, while a control culture was singly infected with wt virus at an MOI of 6.0. After 24 h at 39°C, the cells together with the culture media (3 ml) were harvested and analyzed by plaque assays at 39°C. (B) The experimental protocol is identical to that for panel A, with the exception of the use of a 10-fold lower multiplicity of the wt virus in the double infections and of an MOI of 5.1 in the wt single infection. The infectious virus yields (both assayed at 33°C) obtained 24 h after single infections at the permissive (C) or the nonpermissive (D) temperature, respectively, are shown. Values are means of data from three independent experiments; error bars represent standard deviations.

FIG. 4.

Viral DNA synthesis and virion production of tsOBP mutants after temperature downshift. Multiple Vero cell cultures were infected at an MOI of 10 with tsR or tsS and kept at 39°C for 8 h. Some of the cultures were left at 39°C for a further 16 h, whereas other cultures were transferred to 33°C and harvested at the time points shown. (A) Total DNA was isolated and HSV-specific DNA was quantitated by dot hybridization. (B) The quantity of infectious particles was determined by plaque assay at 33°C. The open triangles (▵) symbolize genome equivalents (panel A) or infectious particles (panel B) from those cells that were kept at the nonpermissive temperature of 39°C and harvested at the shown time points. The open circles (○) symbolize genome equivalents (panel A) or infectious particles (panel B) from those cells that were infected at 39°C, shifted to the permissive temperature, and harvested at the shown time points.