Effects of mutations in the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B on intracellular transport and infectivity

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.     

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34

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-ΔUL34, 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.     

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope–localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside–in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N‐terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer‐mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C‐terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM.     

Antigenic domain 1 is required for oligomerization of human cytomegalovirus glycoprotein B

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant virion envelope protein that has been shown to be essential for the infectivity of HCMV. HCMV gB is also one of the most immunogenic virus-encoded proteins, and a significant fraction of virus neutralizing antibodies are directed at gB. A linear domain of gB designated AD-1 (antigenic domain 1) represents a dominant antibody binding site on this protein. AD-1 from clinical isolates of HCMV exhibits little sequence variation, suggesting that AD-1 plays an essential role in gB structure or function. We investigated this possibility by examining the role of AD-1 in early steps of gB synthesis. Our results from studies using eukaryotic cells indicated that amino acid (aa) 635 of the gB sequence represented the carboxyl-terminal limit of this domain and that deletion of aa 560 to 640 of the gB sequence resulted in loss of AD-1 expression. AD-1 was shown to be required for oligomerization of gB. Mutation of cysteine at either position 573 or 610 in AD-1 resulted in loss of its reactivity with AD-1-specific monoclonal antibodies and gB oligomerization. Infectious virus could not be recovered from HCMV bacterial artificial chromosomes following introduction of these mutations into the HCMV genome, suggesting that AD-1 was an essential structural domain required for gB function in the replicative cycle of HCMV. Sequence alignment of AD-1 with homologous regions of gBs from other herpesviruses demonstrated significant relatedness, raising the possibility that this domain may contribute to multimerization of gBs in other herpesviruses.     

Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.     

Cytoplasmic domain signal sequences that mediate transport of varicella-zoster virus gB from the endoplasmic reticulum to the Golgi

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.     

Mutations in the carboxyl-terminal hydrophobic sequence of human cytomegalovirus glycoprotein B alter transport and protein chaperone binding.

Human cytomegalovirus glycoprotein B (gB) plays a role in the fusion of the virion envelope with the host cell membrane and in syncytium formation in infected cells. Hydrophobic sequences at the carboxyl terminus, amino acids (aa) 714 to 771, anchor gB in the lipid bilayer, but the unusual length of this domain suggests that it may serve another role in gB structure. To explore the function(s) of this region, we deleted aa 717 to 747 (gB deltaI mutation), aa 751 to 771 (gB deltaII mutation), and aa 717 to 772 (gB deltaI-II mutation) and constructed a substitution mutation, Lys-748 to Val (Lys748Val)-Asn749Ala-Pro750Ile (gB KNPm). Mutated forms of gB were expressed in U373 glioblastoma cells and subjected to analysis by flow cytometry, confocal microscopy, and immunoprecipitation. Mutations gB deltaI-II and gB deltaII alone caused secretion of gB into the medium, confirming that aa 751 to 771 function as a membrane anchor. In contrast, mutations gB deltaI and gB KNPm blocked cell surface expression and arrested gB transport in the endoplasmic reticulum (ER). Detailed examination of gB deltaI and gB KNPm with a panel of monoclonal antibodies showed that the mutated forms were indistinguishable from wild-type gB in conformation and formed oligomers; however, they remained sensitive to endoglycosidase H and did not undergo endoproteolytic cleavage. Analysis of protein complexes formed by gB and molecular chaperones in the ER showed that calnexin and calreticulin, lectin-like chaperones, bound equal amounts of uncleaved wild-type gB, gB deltaI, and gB KNPm, but the glucose-regulated proteins 78 (BiP) and 94 formed stable complexes only with the mutated forms, causing their retention in the ER. Our studies show that aa 714 to 750 are key residues in the architecture of gB molecules and that the ER chaperones, which facilitate gB folding and monitor the quality of glycoproteins, detect subtle changes in folding intermediates that are conferred by mutations in this region.     

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.     

Specific induction of Z-DNA conformation by a nuclear localization signal peptide of lupin glutaminyl tRNA synthetase

Recently we have sequenced cDNA of plant glutaminyl-tRNA synthetase (GlnRS) from Lupinus luteus. At the N terminal part the protein contains a lysine rich polypeptide (KPKKKKEK), which is identical to a nuclear localization signal (NLS). In this paper we showed that two synthetic peptides (20 and 8 amino acids long), which were derived from lupin GlnRS containing the NLS sequence interact with DNA, but one of them (8aa long) changing its conformation from the B to the Z form. This observation clearly suggests that the presence of the NLS polypeptide in a leader sequence of GlnRS is required not only for protein transport into nucleus but also for regulation of a gene expression. This is the first report suggesting a role of the NLS signal peptide in structural changes of DNA.     

Identification of a novel signal sequence that targets transmembrane proteins to the nuclear envelope inner membrane

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 beta-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.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best‐characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin‐α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9‐12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.     

Herpes simplex virus type 1 targets the MHC class II processing pathway for immune evasion

HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4(+) T cells is beneficial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4(+) T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression.