Differential Requirements for the Syncytial Phenotype of Herpes Simplex Virus, Type 1
Open Access
- Author:
- Sarfo, Akua Pokuah
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 12, 2017
- Committee Members:
- John Warren Wills, Dissertation Advisor/Co-Advisor
John Warren Wills, Committee Chair/Co-Chair
Neil David Christensen, Committee Member
Jianming Hu, Committee Member
Craig Eugene Cameron, Outside Member - Keywords:
- Herpes Simplex Virus
Glycoproteins
Syncytia
Virology - Abstract:
- HSV-1 encodes four glycoproteins that enable the viral membrane to fuse with the host cell, and this entry complex consists of gD (for receptor-binding), gB (the fusogen), and gH/gL (for signaling between gD and gB). Because this machinery appears on the plasma membrane of infected cells, it must be tightly regulated so that infected cells do not fuse with adjacent uninfected cells. Moreover, the fusion machinery and the proteins that regulate it are also involved in cell-to-cell spread, which enables the virus to move to uninfected cells through lateral junctions so that it can avoid neutralizing antibodies in the host. Syncytial mutants dysregulate this machinery, and cell-cell fusion results in the formation large syncytia, which contain many nuclei bounded by one membrane. Syn variants arise from mutations in any one of four HSV-1 genes, and these include gB, gK, UL20, and UL24. However, the Syn phenotype produced by these mutants is also regulated and requires “accessory” proteins that are involved in regulating the viral entry machinery. It has been assumed that an accessory protein required for one syn locus would be required across all the loci, and limited data support this. For example, it has been reported that accessory protein UL11 is required for both gBSyn and gKSyn. This dissertation project began with the observation that tegument protein UL21 is required for gBSyn. That is, deletion of the UL21-coding sequence from the gBSyn parent resulted in a virus that was lytic even though the syn mutation was present. Because of the assumption that accessory proteins required for one syn locus would be needed for all four, and because the UL21-coding sequence had been completely deleted, we were surprised to see syncytial variants arising from ΔUL21/gBSyn. Similarly, passage of a virus lacking UL21 in an otherwise wild-type background also resulted in the appearance of syncytial variant viruses. We purified and sequenced these ΔUL21 syncytial variants and found that all these mapped exclusively to glycoprotein K, a virion membrane protein capable of binding gB and thought to modulate membrane fusion events. A number of the changes we found were not gKSyn alleles described in the literature, so we followed up by creating recombinant viruses with these changes to confirm that they were indeed novel gKSyn alleles. We also confirmed that UL21 is not necessary for gKSyn-mediated fusion by building recombinant viruses with gK syncytial substitutions lacking UL21, which were immediately fusogenic following viral DNA transfection into Vero cells. Because a differential requirement for an accessory protein in syncytia formation had not been described in the literature, we tested additional gBSyn alleles for their requirement for UL21, as well as testing the requirement for UL21 in the context of UL20 and UL24 syncytial mutations. These studies revealed that UL21 is only required for gBSyn and not for any of the other syn loci. The viral proteins required for syncytia formation are thought to be similar to those needed for direct cell-to-cell spread. Herpesviruses use this spreading mechanism to avoid antibodies that could inactivate it within the infected host. To test whether UL21 is a component of the cell-to-cell spreading machinery, we examined the ability of the null virus to infect neighboring cells in the presence of neutralizing antibodies. For this, no syn mutations were present. Unlike the wild-type control, ΔUL21 virus was found to be greatly impaired in its ability to infect adjacent cells. We hypothesize that this spreading defect provides a selective pressure for syn mutations, which compensate for the cell-to-cell spreading defect. Having identified the first example of a differential requirement for the Syn phenotype, we tested two viral proteins, gE and gI, that are well known to be required for cell-to-cell spread and the gBSyn phenotype. We tested the requirement for these two glycoproteins across the gK, UL20, and UL24Syn backgrounds. They seemed to be the logical next Syn accessory proteins to test because UL21, along with tegument proteins UL11 and UL16 forms a complex on the gE cytoplasmic tail, which is known to modulate gB function in the context of the syncytial mutation gB.A855V. We hypothesized that the requirements for gE and gI would be the same because these two proteins form a heterodimer, and based on the UL21 results, we hypothesized that neither of these glycoproteins would be required for gKSyn, UL20Syn, or UL24Syn. We were surprised to find that gE and gI are differentially required both across and within the syn loci, with UL24 syncytial mutations exhibiting a strict requirement for both gE and gI, UL20 syncytial mutations displaying a strict requirement for gE but not gI, and gK syncytial mutations displaying no requirement for gI, but an allele-specific requirement for gE. The differential requirement for gE within the gKSyn locus is the first example for an accessory protein being differentially required within one syn locus. In the course of our experiments, we also found evidence that additional syn loci might exist. This came from studies of a mutant virus that 1) lacks the UL11-binding site within the cytoplasmic tail of gE and 2) also has a gBSyn mutation (gEΔCT(25)/gB.A855V). This virus was initially lytic when the DNA was transfected into cells; however, variants quickly arose that had the ability to fuse cells. We purified several independent variants, and confirmed that the two original mutations were present. Then, we hypothesized that the variants had gained a second syn mutation. Surprisingly, sequencing of the other three syn loci in these variants revealed no mutations. Because we could not predict what change(s) had occurred, a whole-genome, deep sequencing approach was used. The results revealed that all had mutations that prevented the expression of glycoprotein C, a viral protein implicated in adsorption of the virus to cells but also described in the literature as decidedly not a syn locus. This provides the first evidence that gC regulates the fusion activity of gB.