Identification of a Novel Signal Sequence That Targets Transmembrane Proteins to the Nuclear Envelope Inner Membrane

Abstract

Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble ␤-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885-890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins.The nuclear envelope consists of three morphologically distinct membrane domains (1, 2): (i) the outer nuclear membrane, which is contiguous with the membranes of the rough endoplasmic reticulum (RER), 1 (ii) the pore membranes, which connect outer and inner nuclear membranes and are associated with the nuclear pore complexes, and (iii) the inner nuclear membrane (INM), which is adjacent to the nuclear lamina, a mesh-work of intermediate filament proteins termed lamins (3). Inner and outer nuclear membranes enclose the perinuclear space that continues into that of the RER. While the outer nuclear membrane shares the membrane proteins of the RER, the INM contains a distinct set of integral proteins (4), including the lamin B receptor (LBR) (5) and the lamina-associated polypeptides 1 and 2 (LAP1 and LAP2) (6, 7).In cells that are virus-infected, the various cellular membranes are decorated with specific viral proteins in addition. In the case of herpesviruses that undergo a characteristic nuclear phase of maturation, viral transmembrane glycoproteins are also translocated from the site of their synthesis into the INM, e.g. the glycoprotein B (gB) homologues of Epstein-Barr virus (8, 9), herpes simplex virus type 1 (HSV-1) (10), and human cytomegalovirus (HCMV) (11). The specific localization in the INM not only suggests a role of these proteins in herpesvirus envelopment at the inner nuclear membrane (12) but also implies that specific mechanisms mediate their transport from the RER to the INM compartment. It is currently not precisely known how this apparently specific targeting of cellular and viral transmembrane protein...

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

“…The deduced amino acid sequence does not contain a typical nuclear localization motif (NLS) (24) but contains an RXR (RQR) sequence at amino acids 173 to 175 which has been defined as an efficient INM-sorting motif for human cytomegalovirus glycoprotein B (41,42). However, the significance of this motif in PrV pUL34 has not been investigated, and pUL34 might be small enough to diffuse passively into the INM.…”

“…(23,56), suggesting that nuclear interaction partners arrest the laterally diffusing membrane proteins. However, as a general mechanism, this model has been challenged by the observation that energydependent and receptor-mediated targeting mechanisms exist using either classical nuclear localization signals or INM-specific motifs (5,27,36,41,47).…”

Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

“…The full-length gB that was retained in cells (Fig. 4A) may represent a distinct pool, for example, one targeted to the inner nuclear membrane (17). The fact that the bulk of the MHV-68 gB was processed and incorporated into virions very similarly to the gBs of Kaposi's sarcoma-associated herpesvirus (2) and human cytomegalovirus (33) suggests that is likely to have a similar role in cell binding and entry.…”

The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import

“…At present, it is unclear whether a general mechanism exists for INM targeting, but it is highly suggestive that the diverse INM proteins do not follow a common pathway. First, regarding specific INM sorting motifs, arginine-rich sequences (R-X-R) were identified which suggestively contribute to the ER membrane and nuclear envelope localization of other herpesvirus-encoded membrane proteins (Lee, 1999;Meyer & Radsak, 2000). Similar sequences could principally be found in HCMV pUL50 but apparently do not fulfil the requirements of a potential INM sorting motif (R-H/L/S-R) (Meyer et al, 2002;Shikano & Li, 2003).…”