Efficient quiescent infection of normal human diploid fibroblasts with wild-type herpes simplex virus type 1

Quiescent infection of cultured cells with herpes simplex virus type 1 (HSV-1) provides an important, amenable means of studying the molecular mechanics of a nonproductive state that mimics key aspects of in vivo latency. To date, establishing high-multiplicity nonproductive infection of human cells with wild-type HSV-1 has proven challenging. Here, we describe simple culture conditions that established a cell state in normal human diploid fibroblasts that supported efficient quiescent infection using wild-type virus and exhibited many important properties of the in vivo latent state. Despite the efficient production of immediate early (IE) proteins ICP4 and ICP22, the latter remained unprocessed, and viral late gene products were only transiently and inefficiently produced. This low level of virus activity in cultures was rapidly suppressed as the nonproductive state was established. Entry into quiescence was associated with inefficient production of the viral trans-activating protein ICP0, and the accumulation of enlarged nuclear PML structures normally dispersed during productive infection. Lytic replication was rapidly and efficiently restored by exogenous expression of HSV-1 ICP0. These findings are in agreement with previous models in which quiescence was established with HSV mutants disrupted in their expression of IE gene products that included ICP0 and, importantly, provide a means to study cellular mechanisms that repress wild-type viral functions to prevent productive replication. We discuss this model in relation to existing systems and its potential as a simple tool to study the molecular mechanisms of quiescent infection in human cells using wild-type HSV-1.     

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.