Serial passage is a time-honored viral laboratory method that has been used to select progeny with attenuating mutations (e.g. OKA vaccine strain from varicella zoster virus). In recent years, serial passaging has been used as a method to identify secondary mutations in attenuated HSV oncolytic vectors that mediate improved viral replication and anti-tumor activity. Additional uses for the serial passaging techniqueinclude: i) the testing of genetic stability of biologic therapeutics and potential pathogenic mutations; ii) identification of cryptic gene functions that may have served evolutionarily distant functions for the virus; and iii) to identify antiviral escape mutations, that can then provide leads to improved drug design.

This review focuses on the interferon-induced, innate antiviral response triggered during viral transcription that limits protein synthesis initiation in the infected cell. In addition to discussing the protein product encoded by the HSV γ₁34.5 gene that counters this host antiviral response, the review also describes how mutant viruses lacking the γ₁34.5 gene (Δγ₁34.5) have been studied as vaccine and anti-tumor therapies because of their safety in humans. While these vectors are safe, their limited late viral protein synthesis and diminished replication limit their efficacy. Serial passage of Δγ₁34.5 recombinants has been used to identify whether compensatory mutations improve viral replication or the anti-tumor properties of the Δγ₁34.5 mutants. Current efforts to improve these vectors’ replication are now focused upon engineering a new generation of HSV recombinants based on the earlier serial passage experiments.

Eukaryotic cells contain innate defenses that target viral infection [1]. A principal component of this defense is protein kinase R (PKR), which limits viral gene expression and replication in human cells (summarized in Figure 1) [2].

Low levels of this evolutionarily conserved, interferon-inducible kinase are present in unstressed cells. However, its production is induced by type I interferons or double-stranded RNA (dsRNA) produced during viral infection [1]. Upon binding dsRNA, the kinase activates and phosphorylates itself as well as cellular proteins involved in the antiviral response. The best characterized of the substrates is the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) [3,4]. Phosphorylation of eIF-2α prevents recycling of a critical translation initiation factor, thus limiting viral and cellular protein synthesis in the infected cell [2].

In addition to its antiviral functions, the PKR-mediated host protein shutoff response is also involved in cellular homeostasis. Consequently, second messenger signaling pathways and PKR modulate one another’s activity in the cell. These pathways can block PKR activation during periods of cellular stress or when the cell is actively replicating. For example, upregulation of mitogen-activated protein kinase (MAPK) activity in the cell (mediated by a component of this pathway, MEK) blocks PKR activation during growth factor stimulation [5,6]. Likewise, PKR has also been shown to act as a signal integration point between signaling pathways [7]. While regulation of protein synthesis initiation is the best characterized of the PKR antiviral functions, the kinase also modulates other cellular functions, including: bulk protein degradation in the cell (also called autophagy), RNA transcription, and signal transduction in the cell [5,8-10].

The HSV-1 γ₁34.5 gene encodes a multifunctional protein called ICP34.5 [11,12]. One function, encoded within the 3’ gene domain, blocks host PKR-mediated protein shutoff during infection thus allowing continued late viral protein synthesis in infected cells [13]. During infection, wild-type HSV-1 produces complementary mRNA transcripts that anneal, forming stable dsRNA, which triggers the dimerization and activation of dsRNA-activated host PKR. The ICP34.5 overcomes this PKR-mediated host protein shutoff by binding and recruiting a host phosphatase that specifically dephosphorylates eIF‑2α, allowing continued viral protein synthesis (hereafter referred to as the HSV wild-type protein synthesis phenotype) in the infected cell  (summarized in Figure 1) [14,15].

Recombinant viruses that lack the γ₁34.5 gene, (Δγ₁34.5 HSVs) are incapable of maintaining eIF‑2α in an unphosphorylated form and therefore are unable to maintain protein synthesis in the infected cell [16]. Cessation of protein synthesis occurs at the onset of viral DNA synthesis late in infection, essentially eliminating bulk synthesis of viral structural proteins necessary for viral capsid formation [16]. In addition to structural proteins, viral encapsidation, envelopment and maturation are necessary for efficient viral replication. There are examples of Δγ₁34.5 mutants that are capable of late viral protein synthesis but that cannot negotiate the other steps necessary for viral replication [17]. Evidence suggests that in addition to protein translation, other cellular processes are disrupted in Δγ₁34.5 infected cells that are critical for viral egress. Consequently, Δγ₁34.5 HSV replicate inefficiently and produce fewer progeny virions in cells with intact PKR pathways [18]. While the γ₁34.5 gene product does not interrupt IFN signaling in the infected cell as has been described with the HSV U_L13 and U_L39 gene products, ICP34.5 is critical for HSV evasion of the IFN inducible gene product, PKR. Consequently, Δγ₁34.5 viruses are highly sensitive to Type I IFN and replicate poorly in vivo or in IFN treated cells [19,20]. Restoration of the protein synthesis function allows Δγ₁34.5 HSV to replicate efficiently in the presence of the type l IFN (α or β interferon) response [17].

The γ₁34.5 gene also encodes a second function, neurovirulence, enabling efficient viral replication in post-mitotic neuronal cells [12-14,21]. The neurovirulence and protein synthesis functions encoded by the γ₁34.5 gene are discrete and separable. Late viral protein synthesis can be selectively restored without restoring wild-type neurovirulence [17,22,23]. The Δγ₁34.5 HSV are incapable of efficient replication after direct inoculation in the CNS and do not cause encephalitis [12]. As such, Δγ₁34.5 HSV vectors have been developed as anti-tumor agents for CNS-based malignancies. Whereas 50-100 PFU of wildtype HSV will lead to encephalitis and death in half of the mice inoculated intracerebrally, more than 1 x 10⁷ PFU are required to produce encephalitis and death with a Δγ₁34.5 HSV recombinant [12]. Finally, the γ₁34.5 gene also allows HSV-1 to block autophagy in infected cells [24]. This function contributes to HSV neurovirulence, maps within the 5’ domain of the gene and allows binding and sequestering of beclin-1, a host protein necessary for autophagosome formation [24].

Genetically modified HSV are attractive as replication-competent, oncolytic vectors as well as vaccine vectors for a number of reasons: 1) procedures for constructing novel HSV are well established; 2) genetic modifications (insertions or deletions) do not significantly affect virus replication; 3) considerable experience with the biology of HSV and its behavior in humans and nonhuman primates exists; and 4) modified herpesviruses retain sensitivity to standard antiviral drug therapy as a “built-in” safety feature [25-27]. Deletion of the HSV-1 neurovirulence gene, γ₁34.5, allows the safe administration of recombinant vectors intracranially (for brain tumor therapy) or systemically (for peripheral tumors or as a vaccine vector).

In addition to inhibiting protein synthesis initiation, other viral functions are inhibited as a consequence of this deletional mutation. The Δγ₁34.5 mutants also demonstrate changes in glycoprotein processing Figure 2). The HSV γ₁34.5 gene product differs in amino acid sequence between viral strains. Adoptive transfer studies have shown that these ICP34.5 amino acid differences affect glycoprotein processing and plaque phenotype in cell culture [28,29]. In viruses lacking the γ₁34.5 gene, the differences in glycoprotein processing are even more dramatic. Immunostaining studies show that gD in the Δγ₁34.5 infected cells exist in a single form after separation using denaturing conditions. In contrast, viruses capable of PKR evasion and late viral protein synthesis exhibit multiple slower migratory forms of the glycoprotein indicative of further glycoprotein processing. In Figure 2 the chimeric HSV, C130, which contains the HCMV PKR-evasion gene TRS1, contains multiple forms of glycoprotein D, similar to that observed for wild-type HSV. Confocal immunofluorescence microscopy further demonstrates differences in glycoprotein trafficking in the infected cell. In tumor cells infected with recombinants capable of late viral protein synthesis, gD accumulates within the trans golgi network (TGN) and late endosomal compartment based upon gD and AP-1 staining and colocalization. In contrast, gD expressed in the Δγ₁34.5 infected cells does not localize with adaptor protein complex 1 (AP-1) in the TGN. There are also fundamental differences in the appearance of the TGN. In the Δγ₁34.5 infected cells, the TGN appears disrupted throughout the cytoplasm and does not exhibit the more structured perinuclear aggregates seen in cells capable of continued protein synthesis. The Δγ₁34.5 recombinants also exhibit differences in protein degradation or autophagy within the infected cell [8]. Improved protein production and processing may may enhance the antitumor capabilities of Δγ₁34.5 mutants either directly (improved antigen expression and replication) or indirectly by improving the expression and processing of foreign gene inserts in recombinants for gene therapy applications.

In addition to protein processing recent studies also show that the γ₁34.5 gene product also inhibits IFN response in the cell. ICP34.5 targets the ability of the TANK-binding kinase 1 (TBK1) to activate an antiviral response through interferon regulatory factor 3 (IRF3) and cytokine expression. [30] Phosphorylation and transnuclear translocation of IRF3 as well as expression of downstream genes functioning in antiviral responses are thereby inhibited by the γ₁34.5 protein product. Suppression of the interferon signaling pathway has consistently been shown to be a major mode of the interaction of HSV with the innate immune system, and its interaction with TBK1 appears to be a novel but critical mechanism to ensure successful and productive HSV infection.

The serial passaging of many viruses has resulted in selection of novel mutations providing unique insights into the complexity of evasion of PKR by HSV vectors. The findings of these studies have helped investigators specifically engineer viruses with the goal of improving their anti-tumor activity and producing more effective vaccine vectors. Additionally, other strategies have been investigated which involve the engineering of novel viruses to enhance the oncolytic activity of Δγ₁34.5 HSV oncolytic vectors. These are summarized in the following subsections.

Viral protein synthesis is critical for efficient viral replication. As a consequence, HSV has evolved numerous genes involved in maintaining cellular functions necessary for viral replication. Despite the extensive viral control of the cellular environment, intrinsic intracellular defense systems are nevertheless active and can disrupt viral gene expression. Efforts over the last decade have translated some of the principal work involving HSV innate immune evasion toward development of HSV-1 viral vectors for use as both vaccine and anti-tumor therapy. Fundamental scientific research elucidating these viral mechanisms has made these clinical applications possible.