The Herpes Simplex Virus Type 1 U
            <sub>L</sub>
            17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20 and UL24 To test whether UL21 is needed only for the A855V mutant, additional gB derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gB phenotype. This is the first example of a differential requirement for a viral protein across the four loci. UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

Section: Generation Of a U L 21 Deletion Virusmentioning

confidence: 99%

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype

Sarfo

Starkey

Mellinger

et al. 2017

“…In line with this, a PrV mutant lacking pUL25 forms mature capsids which accumulate in the nucleus in close apposition to the INM but are unable to bud (11). The role of the pUL17 component of the CVSC during nuclear egress is difficult to assess since it is also essential for the mechanistically coupled DNA-encapsidation process and its absence results in the formation of only immature B capsids which contain scaffold but lack viral genome and which are only inefficiently exported from the nucleus (12,13).…”

mentioning

confidence: 99%

Lysine 242 within Helix 10 of the Pseudorabies Virus Nuclear Egress Complex pUL31 Component Is Critical for Primary Envelopment of Nucleocapsids

Rönfeldt

Klupp

Franzke

et al. 2017

Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM) as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 were tested for localization, interaction with pUL34 and complementation of PrV-ΔUL31. Here, we identify a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle. Vesicular transport through the nuclear envelope is a focus of research but still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently providing in-depth molecular details. However, how cargo is incorporated remained unclear. Here, we identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.

“…Structural instability of capsids due to absent or reduced UL25 on capsids also appeared to change the property(ies) of capsids. In support of this suggestion, the absence or reduction in incorporation of UL25 into capsids, which can be caused by the UL25, UL17, and VP26 null mutations, produced improper localization of multiple capsid proteins (e.g., UL17, UL25, VP5, and VP22a) in discrete nuclear domains and appeared to induce aggregation of nuclear capsids (6,19,21,22,43). Previous studies were not able to determine whether the discrete nuclear domains with accumulations of multiple capsid proteins detected by immunofluorescence represent capsid aggregates or are aberrant accumulations of capsid proteins that have not formed capsid structures (6,19,21,22,43).…”

Section: Roles Of Hsv-1 Vp26 In Nucleocapsid Maturationmentioning

confidence: 85%

“…Journal of Virology the DNA concatemers were not cleaved, and only type B capsids were formed (6,(35)(36)(37)(38)(39)(40). Also, mutations in UL25 that preclude its binding to capsids were shown to exhibit the same phenotype as the UL25 null mutation (9), suggesting that UL25 functions in HSV-1 DNA genome packaging in capsids.…”

Section: Roles Of Hsv-1 Vp26 In Nucleocapsid Maturationmentioning

confidence: 99%

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Herpes Simplex Virus 1 Small Capsomere-Interacting Protein VP26 Regulates Nucleocapsid Maturation

Kobayashi

Sagara

Watanabe

et al. 2017

VP26 is a herpes simplex virus 1 (HSV-1) small capsomere-interacting protein. In this study, we investigated the function of VP26 in HSV-1-infected cells with the following results. (i) The VP26 null mutation significantly impaired incorporation of minor capsid protein UL25 into nucleocapsids (type C capsids) in the nucleus. (ii) The VP26 mutation caused improper localization of UL25 in discrete punctate domains containing multiple capsid proteins (e.g., the VP5 major capsid protein) in the nucleus; these domains corresponded to capsid aggregates. (iii) The VP26 mutation significantly impaired packaging of replicated viral DNA genomes into capsids but had no effect on viral DNA concatemer cleavage. (iv) The VP26 mutation reduced the frequency of type C capsids, which contain viral DNA but not scaffolding proteins, and produced an accumulation of type A capsids, which lack both viral DNA and scaffold proteins, and had no effect on accumulation of type B capsids, which lack viral DNA but retain cleaved scaffold proteins. Collectively, these results indicated that VP26 was required for efficient viral DNA packaging and proper localization of nuclear capsids. The phenotype of the VP26 null mutation was similar to that reported previously of the UL25 null mutation and of UL25 mutations that preclude UL25 binding to capsids. Thus, VP26 appeared to regulate nucleocapsid maturation by promoting incorporation of UL25 into capsids, which is likely to be required for proper capsid nuclear localization. HSV-1 VP26 has been reported to be important for viral replication and virulence in cell cultures and/or mouse models. However, little is known about the function of VP26 during HSV-1 replication, in particular, in viral nucleocapsid maturation although HSV-1 nucleocapsids are estimated to contain 900 copies of VP26. In this study, we present data suggesting that VP26 promoted packaging of HSV-1 DNA genomes into capsids by regulating incorporation of capsid protein UL25 into capsids, which was reported to increase stability of the capsid structure. We also showed that VP26 was required for proper localization of capsids in the infected cell nucleus. This is the first report showing that HSV-1 VP26 is a regulator for nucleocapsid maturation.