Investigating the role of the inner tegument proteins in HSV-1 infection.
PhD thesis, University of Glasgow.
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Extensive study of the prototypical alphaherpesvirus, Herpes simplex virus type-1 (HSV-1), has revealed much about its biology and structure. The characteristic virion comprises a DNA containing capsid surrounded by consecutive layers of proteinaceous tegument and lipid envelope. Our understanding of the capsid is relatively advanced compared to that of the tegument. The tegument has previously been thought of as an amorphous accumulation of protein filling the space between the capsid and envelope.
The tegument has been subdivided into the inner and the outer tegument with the proteins pUS3, pUL36 and pUL37 assigned to the inner tegument. A number of studies have pointed to the essential role of both pUL36 and pUL37 in virion morphogenesis by demonstrating a block on secondary envelopment in the absence of either protein. However, the phenotypes attributable to deletion mutants of UL36 and UL37 varied between HSV-1 and the related alphaherpesvirus, Pseudorabies virus (PrV), and between different PrV mutants. In particular the phenotypes of an HSV-1 mutant unable to express functional pUL36 due to an internal deletion between amino acids 362-1555 (KΔUL36) and PrV mutants lacking UL36 showed considerable variation. Both accumulated DNA filled cytoplasmic nucleocapsids, however, those produced by KΔUL36 formed small aggregates within the cytoplasm in contrast to PrV where the capsids were dispersed. UL36 and UL37 deletion mutants were also reported to retain capsids to the nucleus implying a function in nuclear exit.
In order to clarify the seeming disparities between these mutants, novel deletions of both UL36 and UL37 were engineered into HSV-1 17(+). The deletion of UL36 encompassed the entire open reading frame (ORF) while the UL37 deletion removed all but the 3 most C terminal amino acids. Examination by EM revealed that these mutants recapitulate the block on secondary envelopment that is characteristic of inner tegument mutants reported to date, with accumulation of nucleocapsids within the cytoplasm. This examination revealed that the cytoplasmic capsids of the complete UL36 deletion adopted the dispersed phenotype seen in PrV suggesting the phenotype of KΔUL36 is specific to the mutation introduced. Furthermore, this analysis failed to demonstrate a retention of capsids within the nucleus in the absence of either pUL36 of pUL37, indicating that neither has a definitive role in nuclear exit, and the principle function of these proteins is within secondary envelopment.
Work presented within this thesis using IF and Immuno-EM methods shows that pUL36 is associated with the cytoplasmic clusters of capsids produced by a mutant lacking pUL37 suggesting that this may contribute to the aggregation of these capsids. Furthermore, these pUL36-decorated cytoplasmic nucleocapsids appear able to recruit pUL48 but are blocked in secondary envelopment, in contrast to PrV lacking UL37 where virions are formed but at much reduced levels. In the absence of pUL36 no tegument is recruited to cytoplasmic nucleocapsids but both minor capsid associated proteins pUL17 and pUL25 are present.
Cytoplasmic capsids of mutants lacking pUL36 and pUL37 have been isolated and examined biochemically. Cytoplasmic capsids of KΔUL36 were found to be associated with the N terminal fragment (1-361 amino acids) of pUL36 expressed by this virus, implying that a capsid binding activity is present within this region, which is the first demonstration of such an activity outside of the previously mapped activity within the 62 C terminal amino acids of pUL36. The difference in behaviour between KΔUL36 and the complete UL36 deletion also implies that the aggregation of capsids in KΔUL36 is caused by the presence of this fragment.
Assembly of L particles, structures consisting of tegument and envelope but lacking capsids, was unaffected by the deletion of either UL36 or UL37. Biochemical characterisation of purified L particles revealed that deletion of pUL36 prevents the incorporation of pUL37 and deletion of pUL37 leads to the absence of pUL36 despite their expression during infection. This suggests that incorporation of inner tegument to L particles, and envelopment of nucleocapsids, requires pUL36 and pUL37 to be present in a complex. The sole exception to this was the incorporation of the N terminal fragment of pUL36 in KΔUL36 infection into L particles in the absence of pUL37 the mechanism for which is unknown.
Little of the role of the inner tegument proteins in initiating infection has been established. Previous studies have hinted at functions in nuclear pore complex binding and genome release, while a growing body of data implicates pUL36 and pUL37 in cytoplasmic transport of nucleocapsids. As both pUL36 and pUL37 are essential in HSV-1 virion morphogenesis a method was required to establish the defects of inner tegument deletion mutants in the initiation of infection. This was provided by infecting cells at a low multiplicity with mutant viruses (0.01pfu/cell) and subsequently fusing the cells into syncytia, in which several nuclei shared a common cytoplasm. The spread of viral DNA replication in these nuclei was tracked by fluorescence in situ hybridisation. In each syncytium the initially infected nucleus produces nucleocapsids that enter the common syncytial cytoplasm and have access to naïve nuclei without needing to undergo secondary envelopment. Using this system the essential role of pUL36 in the initiation of infection was confirmed and it was also shown that pUL37 is dispensable for the initiation of new cycles of viral DNA replication in naïve nuclei. The behaviour of cytoplasmic nucleocapsids within syncytia seems to be equivalent to that of incoming virion derived tegumented nucleocapsids as WT HSV-1 and HSV-1 ΔUL37 infections behaved similarly and were equally affected by treatment with nocodazole suggesting that both are undertaking similar activities.
The work presented here has helped to clarify the roles of the inner tegument proteins during virus assembly and the methods developed have extended our ability to assess the contributions of proteins essential for virion formation, in the initiation of infection. The studies presented here reinforce the idea that pUL36 may play multiple roles in the initiation of infection.
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