Structural Determinants for Nuclear Envelope Localization and Function of Pseudorabies Virus pUL34

Abstract

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-⌬UL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function. During herpesvirus morphogenesis, nucleocapsids are assembled in the host cell nucleus and have to cross the nuclear membranes to gain access to the cytosol, where final tegumentation and envelopment occurs. To this end, nucleocapsids bud at the inner nuclear membrane (INM), which subsequently encloses the nucleocapsid, thereby forming a primary enveloped virion located in the perinuclear space. This primary envelope is lost after fusion with the outer nuclear membrane (ONM), releasing the nucleocapsid into the cytosol (reviewed in references 25, 38, 39, and 40). To gain access to the budding sites at the INM, the nuclear lamina, a filamentous meshwork consisting mainly of lamin types A/C and B which underlies and supports the nuclear membrane, has to be softened and/or dissolved at least locally (reviewed in references 25 and 40). This partial dissolution is thought to be accomplished by the nuclear egress complex (NEC), which is highly conserved throughout the herpesviruses (reviewed in references 25 and 40). It consists of viral proteins homologous to herpes simplex virus type 1 (HSV-1) pUL34 and pUL31 and functions via the recruitment of cellular and viral protein kinases which phosphorylate lamins, thereby triggering their dissolution (3, 43, 49). In the absence of either pUL31 or pUL34, nucleocap...

A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope

“…When EGFP-pUL31 was added, it was efficiently recruited to the GUV membranes regardless of whether pUL34 was attached to the membrane via its N or C terminus. This finding is consistent with the fact that the transmembrane region of pUL34 is not required for pUL31 interaction (9,38). More importantly, pUL34-mediated pUL31 recruitment was sufficient for intralumenal vesicle formation.…”

Membrane deformation and scission by the HSV-1 nuclear egress complex

“…Yet, we show that the budding of the acidic GUVs by soluble NEC220 is recapitulated by the budding of the less acidic GUVs by the membrane-anchored NEC220-His, which establishes that our in-vitro system adequately models the in-vivo budding and can be used as an experimental system for studying the budding mechanism in vitro . Moreover, although the TM of UL34 is essential in vivo, its substitution with the TMs of other nuclear membrane proteins does not affect the function 26 arguing that the primary role of the TM is to anchor the NEC in the INM rather than to participate in membrane budding directly.…”

Venture from the Interior—Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane

“…Unlike the soluble pUL31, pUL34 is a tail-anchored (TA) membrane protein ( Figure 2 B; [ 17 , 58 ]; and references therein). In all pUL34 orthologs, the cyto-/nucleoplasmically exposed N-terminal domains and the C-terminal TA domains are conserved and essential for viral replication ( Figure 2 B; [ 58 , 59 , 60 ]). The TA can be replaced by alternative anchor domains [ 58 , 60 ] and deletions of large parts of the linker region between the conserved N-terminal domain and the essential TA domain are tolerated [ 61 ].…”