The loss of both pUL16 and pUL21 in HSV-1-infected cells alters capsid–tegument composition, nuclear membrane architecture, cytoplasmic maturation and cell-to-cell spread

Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1) strain KOS. To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation-associated recombination. Using this method, we generated null mutations in five tegument protein-coding genes as well as different combinations of these mutants. The single-locus mutants were all able to plaque on Vero cells. However, one multi-locus combination, ∆UL16/UL21, proved lethal for virus replication in non-permissive cells. The proteins encoded by the genes UL16 and UL21 are of interest because they are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1-infected cells are unclear. Both are dispensable for HSV-1 replication; however, in HSV-2, their mutation results in nuclear retention of assembled capsids and has activities that impact nuclear membrane integrity as well as activities of proteins that function in nuclear egress. We thus characterized these HSV-1 viruses that carry the single and double mutants. What we found was that the single mutants could replicate within cells and spread from infected to uninfected cells, albeit at significantly reduced levels. However, the double mutant (∆16/21) could not produce infectious progeny in a 24 h growth cycle and could not spread from cell to cell. Confocal microscopy of VP16-Venus expressed by these viruses as well as immunofluorescence assays for glycoprotein B showed perturbation of the nuclear membrane, which was pronounced in ∆21 and ∆16/21 infected cells. All the mutants assembled DNA-filled capsids as judged by ultrastructural analyses and sedimentation studies. Electron microscopy revealed the presence of numerous mature viruses in WT-infected cells but fewer such particles in the ∆16- and ∆21-infected cells. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident, but mature enveloped particles were not detected. The capsid particles isolated from all the single- and multi-locus mutant-infected cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the WT capsids. This reduced incorporation may be related to the loss of the integrity of the architecture of the nuclear membrane. Interestingly, the incorporation of pUL16 was not affected by the absence of pUL21 and vice versa, as judged by immunoblots. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16, the complex of pUL16 and pUL21 should be considered as important mediators of maturation and cell-to-cell spread of the particle.