Biology subjects

Roller, R. J.

Publications and source records attributed to Roller, R. J..

4 recordsLinked to original sources

bioRxiv2023

The universal suppressor mutation in the HSV-1 nuclear egress complex restores membrane budding defects by stabilizing the oligomeric lattice

Nuclear egress is an essential process in herpesviral replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress - budding at the inner nuclear membrane - is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because mutations designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Here, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. We demonstrate that the suppressor mutation indirectly promotes the formation of new contacts between the NEC hexamers that, ostensibly, stabilize the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

biophysics↗

bioRxiv2023

DISTINCT ROLES OF VIRAL US3 AND UL13 PROTEIN KINASES IN HERPES VIRUS SIMPLEX TYPE 1 (HSV-1) NUCLEAR EGRESS

Herpesviruses transport nucleocapsids from the nucleus to the cytoplasm by capsid envelopment into the inner nuclear membrane and de-envelopment from the outer nuclear membrane, a process that is coordinated by nuclear egress complex (NEC) proteins, pUL34, and pUL31. Both pUL31 and pUL34 are phosphorylated by the virus-encoded protein kinase, pUS3, and phosphorylation of pUL31 regulates NEC localization at the nuclear rim. pUS3 also controls apoptosis and many other viral and cellular functions in addition to nuclear egress, and the regulation of these various activities in infected cells is not well understood. It has been previously proposed that pUS3 activity is selectively regulated by another viral protein kinase, pUL13 such that its activity in nuclear egress is pUL13-dependent, but apoptosis regulation is not, suggesting that pUL13 might regulate pUS3 activity on specific substrates. We compared HSV-1 UL13 kinase-dead and US3 kinase-dead mutant infections and found that pUL13 kinase activity does not regulate the substrate choice of pUS3 in any defined classes of pUS3 substrates and that pUL13 kinase activity is not important for promoting de-envelopment during nuclear egress. We also find that mutation of all pUL13 phosphorylation motifs in pUS3, individually or in aggregate, does not affect the localization of the NEC, suggesting that pUL13 regulates NEC localization independent of pUS3. Finally, we show that pUL13 co-localizes with pUL31 inside the nucleus in large aggregates, further suggesting a direct effect of pUL13 on the NEC and suggesting a novel mechanism for both UL31 and UL13 in the DNA damage response pathway. IMPORTANCE Herpes simplex virus infections are regulated by two virus-encoded protein kinases, pUS3 and pUL13, which each regulate multiple processes in the infected cell, including capsid transport from the nucleus to the cytoplasm. Regulation of the activity of these kinases on their various substrates is poorly understood, but importantly, kinases are attractive targets for the generation of inhibitors. It has been previously suggested that pUS3 activity on specific substrates is differentially regulated by pUL13 and, specifically, that pUL13 regulates capsid egress from the nucleus by phosphorylation of pUS3. In this study, we determined that pUL13 and pUS3 have different effects on nuclear egress and that pUL13 may interact directly with the nuclear egress apparatus with implications both for virus assembly and egress and, possibly, the host cell DNA- damage response.

microbiology↗

bioRxiv2021

Cell culture evolution of a HSV-1/VZV UL34/ORF24 chimeric virus reveals novel functions for HSV genes in capsid nuclear egress

HSV and VZV are both members of the alphaherpesvirus subfamily, but are of different genera. Substitution of the HSV-1 UL34 coding sequence with that of its VZV homolog, ORF24, results in a virus that has defects in viral growth, spread, capsid egress, and nuclear lamina disruption very similar to those seen in a UL34-null virus despite normal interaction between ORF24 protein and HSV pUL31 and proper localization of the NEC at the nuclear envelope. Minimal selection for growth in cell culture resulted in viruses that grew and spread much more efficiently that the parental chimeric virus. These viruses varied in their ability to support nuclear lamina disruption, normal NEC localization and capsid de-envelopment. Single mutations that suppress the growth defect were mapped to the coding sequences of ORF24, ICP22 and ICP4 and one virus carried single mutations in each of the ICP22 and US3 coding sequences. The phenotypes of these viruses support a role for ICP22 in nuclear lamina disruption and a completely unexpected role for the major transcriptional regulator, ICP4, in capsid nuclear egress. ImportanceInteractions among virus proteins are critical for assembly and egress of virus particles, and such interactions are attractive targets for antiviral therapy. Identification of critical functional interactions can be slow and tedious. Capsid nuclear egress of herpesviruses is a critical event in the assembly and egress pathway and is mediated by two proteins that are conserved among herpesviruses, pUL31 and pUL34. Here we describe a cell culture evolution approach to identify other viral gene products that functionally interact with pUL34.

microbiology↗

bioRxiv2020

HSV Forms an HCMV-like Viral Assembly Center in Neuronal Cells

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is the site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization. SummaryThis study shows that HSV forms a viral assembly center in neuronal cells by reorganization of host membranes. This system is a novel and powerful tool to study herpesvirus assembly pathways and host cell membrane dynamics.

cell biology↗