The nuclear egress complex (NEC) is required for efficient translocation of newly synthesized herpesvirus nucleocapsids from the nucleus to the cytosol. It consists of the type II membrane protein pUL34 which interacts with pUL31 at the inner nuclear membrane (INM). To map regions within pUL34 required for nuclear membrane targeting and pUL31 interaction, we constructed deletion/substitution mutations. Previously, we showed that 50 C-terminal amino acids (aa) of pseudorabies virus (PrV) pUL34, including the transmembrane domain, could be functionally replaced by cellular lamina-associated polypeptide 2␤ (Lap2␤) sequences. In contrast, replacement of the C-terminal 100 aa abrogated complementation but not pUL31 interaction. To further delineate essential sequences within this region, C-terminal pUL34 truncations of 60, 70, 80, 85, and 90 aa fused to Lap2␤ sequences were generated. While truncations up to 85 aa were functional, deletion of the C-terminal 90 aa abrogated function, which indicates that the important region is located between aa 171 and 176. Amino acids 173 to 175 represent RQR, a motif suggested to mediate INM targeting. Mutagenesis to RQG revealed that the mutant protein exhibited pronounced Golgi localization, but a fraction still reached the INM. Deletion mutations in the N-terminal domain of pUL34 demonstrated that absence of the first 4 aa was tolerated, while removal of 9 or more residues resulted in a nonfunctional protein. In addition, mutation of three conserved cysteines did not abrogate pUL34 function, whereas alteration of a conserved glutamine/tyrosine sequence yielded a nonfunctional protein.H erpesvirus capsids are assembled and viral genomes are packaged in the host cell nucleus while further virion maturation occurs in the cytosol. For transit from the nucleus to the cytoplasm, herpesvirus nucleocapsids bud at the inner nuclear membrane (INM), thereby acquiring a primary envelope which subsequently fuses with the outer nuclear membrane (ONM) to release the nucleocapsids into the cytosol. This process can be regarded as a vesicle (primary envelope)-mediated transport of cargo (nucleocapsids) through the nuclear envelope. Although mechanistic details of this process are not yet known, conserved herpesviral proteins homologous to herpes simplex virus 1 (HSV-1) pUL31 and pUL34 are required for efficient translocation (reviewed in references 1 to 3).The pUL34 homologs constitute type II, tail-anchored membrane proteins which are efficiently targeted to the nuclear envelope. The pUL34 interaction partner pUL31 is diffusely distributed in the nucleoplasm in the absence of pUL34 but relocates to the INM in its presence, where both proteins form the heterodimeric nuclear egress complex (NEC). This complex recruits cellular and viral kinases to phosphorylate the nuclear lamina, a tight network of intermediate filaments underneath the INM, to partly dissolve or soften the lamina, and to permit access of nucleocapsids to the budding sites (reviewed in references 1, 3, and 4). Expression of only...

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Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer’s patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host’s intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies.

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...