Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

Herpesviruses morphogenesis occurs stepwise both temporally and spatially, beginning in the nucleus and concluding with the emergence of an extracellular virion. The mechanisms by which these viruses interact with and penetrate the nuclear envelope and subsequent compartments of the secretory pathway remain poorly defined. In this report, a conserved viral protein (VP1/2; pUL36) that directs cytoplasmic stages of egress is identified to have multiple isoforms. Of these, a novel truncated VP1/2 species translocates to the nucleus and assists the transfer of DNA-containing capsids to the cytoplasm. The capsids are handed off to full-length VP1/2, which replaces the nuclear isoform on the capsids and is required for the final cytoplasmic stages of viral particle maturation. These results document that distinct VP1/2 protein species serve as effectors of nuclear and cytoplasmic egress.

Section: Discussionsupporting

confidence: 89%

Nuclear Egress of Pseudorabies Virus Capsids Is Enhanced by a Subspecies of the Large Tegument Protein That Is Lost upon Cytoplasmic Maturation

Leelawong

Lee

Smith

2012

“…In this model, suppressor mutations in any of pUL31 CR1, pUL34 CR3, or pUL34 CR1 might stabilize the pUL34/pUL31 complex, restoring stable pUL31/ pUL34 and perhaps proper regulation of budding. Involvement of the UL34 N terminus in formation and targeting of the NEC to the NE has not previously been reported for HSV UL34 but has been seen with the mouse cytomegalovirus (MCMV) homolog M50, where mutation of the tyrosine at position 57 causes failure of NE localization (4,28). CL13 and cell-cell spread.…”

Section: Discussionmentioning

confidence: 99%

“…Consistent with this, a construct containing CR1, CR2, and most of CR3 (aa 1 to 161) of pseudorabies virus (PRV) pUL34 was sufficient to interact with pUL31 in a yeast two-hybrid assay (10). In mouse cytomegalovirus (MCMV), on the other hand, use of small insertions and point mutations implicated a different region of the UL34 homolog, M50, in binding to the UL31 homolog, M53 (4,19,28). The interaction region is located in a highly conserved stretch of residues at the N terminus of M50 CR2.…”

mentioning

confidence: 99%

Intragenic and Extragenic Suppression of a Mutation in Herpes Simplex Virus 1 UL34 That Affects both Nuclear Envelope Targeting and Membrane Budding

Roller

Haugo

Kopping

2011

Late in infection herpesviruses move DNA-filled capsids from the nucleus to the cytoplasm by enveloping DNA-containing capsids at the inner nuclear membrane (INM) and deenveloping them at the outer nuclear membrane. This process requires two conserved herpesvirus proteins, pUL31 and pUL34. Interaction between pUL34 and pUL31 is essential for targeting both proteins to the nuclear envelope (NE), and sequences that mediate the targeting interaction have been mapped in both proteins. Here, we show that a mutation in the INM-targeting domain of pUL34 fails to support production of infectious virus or plaque formation. The mutation results in multiple defects, including impaired interaction between pUL34 and pUL31, poor NE targeting of pUL34, and misregulated, capsid-independent budding of the NE. The mutant defects in virus production, plaque formation, and pUL31 interaction can be suppressed by other mutations in the INMtargeting domain of pUL31 and by additional mutations in the pUL34 coding sequence.Efficient nuclear egress of herpesviruses requires formation of a nuclear envelopment complex (NEC) consisting of viral and cellular proteins (24,26,34). The NEC contains homologs of the herpes simplex virus (HSV) UL31 and UL34 proteins (called pUL31 and pUL34), and these are critical for nuclear egress in all herpesviruses tested (9,15,24,25,33). pUL31 and pUL34 homologs interact with each other, and formation of a pUL31/pUL34 complex is required for proper targeting of the complex to the nuclear envelope (NE) (10,16,30,31,36,37,41). The interactions that underlie complex formation are therefore critical for assembly and egress of all herpesviruses.Despite the conservation of a pUL31-pUL34 interaction, it is not clear that the structural basis for that interaction is completely conserved. The sequence of pUL31 and its homologs can be divided into four conserved regions (CRs) (Fig. 1B) (37). The most N-terminal of these regions (CR1) has been shown to mediate interaction with pUL34 homologs in examples from all herpesvirus subfamilies (19, 37). The situation with pUL34 homologs is less clear. For HSV-1 pUL34, the sequence that interacts with pUL31 and that is required for nuclear envelope targeting was mapped by deletion and domain swapping to amino acids (aa) 137 to 181 (18). This corresponds to the third of three CRs in the pUL34 sequence (Fig. 1A). Consistent with this, a construct containing CR1, CR2, and most of CR3 (aa 1 to 161) of pseudorabies virus (PRV) pUL34 was sufficient to interact with pUL31 in a yeast two-hybrid assay (10). In mouse cytomegalovirus (MCMV), on the other hand, use of small insertions and point mutations implicated a different region of the UL34 homolog, M50, in binding to the UL31 homolog, M53 (4,19,28). The interaction region is located in a highly conserved stretch of residues at the N terminus of M50 CR2. Whether the differences between MCMV M50 and HSV pUL34 reflect a very different structural basis for interaction is not yet clear.At the NE, pUL34 and pUL31 mediate subsequent steps ...

“…The HCMV NEC is also required for nuclear lamina disruption, and it recruits UL97 to the nuclear rim (8), suggesting that this NEC-UL97 association is important for lamin phosphorylation and nuclear lamina dissolution by UL97. Based on work with other herpesviruses, the NEC is also thought to orchestrate primary envelopment and possibly other steps in nuclear egress (29)(30)(31).…”

mentioning

confidence: 99%

Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Sharma

Bender

Kamil

et al. 2015

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses. Herpesviruses replicate and package their DNA genomes into capsids in the nucleus of the host cell. The nucleocapsids are then transported out of the nucleus through an unusual process called nuclear egress. A widely accepted model for nuclear egress entails envelopment and de-envelopment of capsids as they transit the nuclear membranes (reviewed in references 1, 2, and 3). Studies of human cytomegalovirus (HCMV) nuclear egress are of particular interest, because of the medical ...