Relationship of herpes simplex virus genome configuration to productive and persistent infections

Abstract

Infection of susceptible cells by herpes simplex virus (HSV) can lead to productive infection or to latency, where the genomes persist in the nuclei of peripheral neurons in a quiescent state. Using the HSV strain d109, which does not express any viral genes and thus establishes a quiescent state in most cells, we observed that a fraction of genomes circularized upon infection. The expression of infected cell protein (ICP) 0, which is known to be involved in reactivation from latency and the promotion of productive infection, inhibited the formation of circular genomes. Circular genomes were not observed upon infection of fully permissive cells by wild-type virus, in either the presence or absence of viral DNA replication. However, productive infection in the absence of ICP0 resulted in the accumulation of a subpopulation of circular genomes. The proportion of circular genomes formed during infection with an ICP0 mutant was greater at low multiplicity of infection, a condition in which ICP0 mutants replicate poorly. In the complete absence of viral gene expression, it was found that only circular genomes persisted in cells. These results suggest that circularization of the HSV genome may not occur early in the productive phase of wild-type HSV infection, but rather during establishment of a quiescent state or latency, providing a possible strategy for long-term persistence. Additionally, the circularization and possible fate of HSV genomes are regulated by an activity of ICP0.

Fig. 1.

Gardella gel analysis of d109- and d106-infected cells. (A) Monolayers of Vero cells were infected at a moi of 20 PFU per cell and incubated for the indicated times before analysis as described in Materials and Methods.(B) Vero cells were infected with d109 or d106 at a moi of 20 PFU per cell for 24 h. Nuclei were then isolated and run on the gels as described in Materials and Methods. In both A and B, the mobility of linear viral DNA is indicated by V and the low-mobility band is indicated by an arrow. The mobility of linear viral DNA was determined by examining the migration rate of virion DNA in samples where the virus was adsorbed to cells on ice (0 h; A), or where mock-infected cells were spiked with 5 × 10⁶ PFU of d109 or d106 virions (B).

Fig. 2.

Characterization of the low-mobility form of the d109 genome. (A) d109 virions (lane 1), d109-infected cells (lane 2), and Raji cells (lane 3) were analyzed on a Gardella gel and probed for HSV and EBV genomes. (B) Template for eluting regions a, b, c, and d from a Gardella gel of d109 virions (lane 4) and d109-infected cells (lane 5). (C) The DNA in samples a–d in B were analyzed as described in Materials and Methods to observe the abundance of joint (J) and terminal (T) fragments. Different amounts of d were loaded on the gel. (D) Vero cells were infected at the indicated moi of d109 for 24 h, run on a Gardella gel, subjected to Southern blot analysis, and quantified. (E) The kinetics of circularization of d109 genomes in Vero cells was determined as described in the text.

Fig. 4.

Circularization of the HSV genome during productive infection in the presence and absence of ICP0. Vero cells were infected with wt virus (strain KOS) or d99 and incubated in the presence or absence of PAA for the indicated times before gel analysis. d109-infected cells were also included on all of the gels. (A) moi of 20 PFU per cell. (B) moi of 1 PFU per cell. (C) Overexposure of the d99 gel in A.(D) Vero cells were infected at a moi of 3 PFU per cell of the indicated virus and incubated for 6 h in the presence of PAA before gel analysis.

Fig. 3.

ICP0 expression from adenovirus inhibits circularization of d109 in trans. (A) Phase-contrast and EGFP fluorescence images of Vero cells infected with d109. Cells infected with d109 were superinfected with AdS.11D (Ad.) or AdS.11E4(ICP0) (Ad.ICP0) at a moi of ≈50 PFU per cell directly postinfection with d109 where indicated. The infected cultures were analyzed 24 h postinfection. (B) d109-infected cells were superinfected with the adenoviruses as described above and analyzed on Gardella gels.

Fig. 5.

Stability of circular and linear d109 genomes during long-term persistent infection. Vero cells were infected with d109 at 20 PFU per cell and incubated for the indicated time before analysis, as described in Materials and Methods.