Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex

Paßvogel, Lars; Trübe, Patricia; Schuster, Franziska; Klupp, Barbara G.; Mettenleiter, Thomas C.

doi:10.1128/jvi.00021-13

Cited by 20 publications

(38 citation statements)

References 49 publications

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“…2 C and D) all decreased affinity by at least fivefold. This segment of UL50 and M50, particularly E56 and Y57, and the corresponding segment of their pseudorabies virus and Kaposi's sarcoma herpes virus homologs have previously been shown to be important for interaction with the other NEC subunit in less quantitative coimmunoprecipitation and colocalization assays (16,19,32,33). We also found that several substitutions that alter residues between positions 121 and 129 of M50 and 125 and 139 of UL50 (at the top of the groove and within the cavity in Fig.…”

Section: Significancesupporting

confidence: 52%

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Leigh

Sharma

Mansueto

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.H erpesviruses encompass a large family of infectious agents, including important veterinary and human pathogens (1). Among the latter is human cytomegalovirus (HCMV), which can cause serious disease, particularly in immunocompromised individuals and newborns (2). Despite the importance of HCMV in these medically vulnerable populations, currently available treatment options suffer from issues with toxicities, drug resistance, and/or pharmacokinetics (2, 3), motivating the identification of new drug targets.All herpesviruses of mammals, birds, and reptiles undergo a remarkable process known as nuclear egress as part of the viral lifecycle. It is generally accepted that, after assembly in the nucleus, the viral nucleocapsid undergoes envelopment to cross the inner nuclear membrane (INM) followed by deenvelopment to cross the outer nuclear membrane, resulting in release into the cytoplasm for continuation of the virion maturation process (4). Nuclear egress is orchestrated by a highly conserved, heterodimeric nuclear egress complex (NEC), which recruits one or more protein kinases to disrupt the nuclear lamina, permitting access of nucleocapsids to the INM, where the NEC induces budding of the nucleocapsid into the perinuclear space (5-13). In HCMV, the NEC is comprised of UL50, which is an INM protein, and UL53, which is a nucleoplasmic protein that is brought to the INM by its interaction with UL50. These two proteins and their murine CMV (MCMV) homologues, M50 and M53, are essential for replication and nuclear egress (8, 14-17) of ...

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Section: Significancesupporting

confidence: 52%

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Leigh

Sharma

Mansueto

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the spatiotemporal regulation of NEC formation and function and how nucleocapsids are recruited into these vesicles remain incompletely understood. Mutational studies of pUL34 have already shed some light on the mechanism of NEC formation and function (12,(18)(19)(20)(21)(22)(23), but corresponding data on pUL31 homologs are less comprehensive.…”

Section: Discussionmentioning

confidence: 99%

“…For complementation studies, cell lines stably expressing the pUL31-NLS and pUL31-NES mutant constructs were generated. To this end, RK13 cells were transfected with the mutated pcDNA-UL31 plasmids by calcium phosphate precipitation (19,33). Cells were selected in medium containing 0.5 mg/ml G418, and correct protein expression was verified by indirect immunofluorescence and Western blot analysis.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…However, the molecular details of this interaction are still not fully understood and no crystal structures of the NEC or of the single proteins are available yet. Deletion and substitution analyses for different pUL34 homologs showed that the N-terminal part, which is well conserved between the different homologs, is necessary for pUL31 binding while the C terminus comprising the transmembrane domain is required for correct positioning but can be functionally substituted by heterologous sequences as long as a membrane anchor is provided (12,(18)(19)(20)(21)(22)(23).…”

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confidence: 99%

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Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

Paßvogel

Klupp

Granzow

et al. 2015

J Virol

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The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-⌬NLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

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“…To introduce mutations into the gH sequence, a QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) was used as described recently (28). Specific primers (Table 1) derived from the genomic sequence of PrV-Ka (GenBank accession no.…”

Section: Cells and Virusesmentioning

confidence: 99%

The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion

Schröter

Klupp

Fuchs

et al. 2014

J Virol

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Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-prolinecysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline 438 induces bending at the end of an alpha-helix, thereby placing cysteine 404 and cysteine 439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine 438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline 438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine 404 to serine. Although PrV expressing gHC 404 S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine 438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59. IMPORTANCEThe role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline 438 in PrV gH to allow the formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions: mutation of this residue resulted in a drastic impairment of membrane fusion kinetics not only in vitro in transient transfection-fusion assays but also during virus infection. Elimination of formation of the disulfide bond yielded the same phenotype in transient assays but had a more drastic effect on virus replication. Thus, our studies add important information to structure-function analyses of herpesvirus gH.

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Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex

Cited by 20 publications

References 49 publications

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion

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