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Productive viral infection often depends on the manipulation of the cytoskeleton. Herpesviruses, including rhesus monkey rhadinovirus (RRV) and its close homolog, the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8), exploit microtubule (MT)-based retrograde transport to deliver their genomes to the nucleus. Subsequently, during the lytic phase of the life cycle, the maturing viral particles undergo orchestrated translocation to specialized regions within the cytoplasm, leading to tegumentation, secondary envelopment, and then egress. As a result, we hypothesized that RRV might induce changes in the cytoskeleton at both early and late stages of infection. Using confocal imaging, we found that RRV infection led to the thickening and acetylation of MTs emanating from the MT-organizing center (MTOC) shortly after viral entry and more pronounced and diffuse MT reorganization during peak stages of lytic gene expression and virion production. We subsequently identified open reading frame 52 (ORF52), a multifunctional and abundant tegument protein, as being the only virally encoded component responsible for these cytoskeletal changes. Mutational and modeling analyses indicated that an evolutionarily conserved, truncated leucine zipper motif near the N terminus as well as a strictly conserved arginine residue toward the C terminus of ORF52 play critical roles in its ability to rearrange the architecture of the MT cytoskeleton. Taken together, our findings combined with data from previous studies describing diverse roles for ORF52 suggest that it likely binds to different cellular components, thereby allowing context-dependent modulation of function. IMPORTANCE A thorough understanding of the processes governing viral infection includes knowledge of how viruses manipulate their intracellular milieu, including the cytoskeleton. Altering the dynamics of actin or MT polymerization, for example, is a common strategy employed by viruses to ensure efficient entry, maturation, and egress as well as the avoidance of antiviral defenses through the sequestration of key cellular factors. We found that infection with RRV, a homolog of the human pathogen KSHV, led to perinuclear wrapping by acetylated MT bundles and identified ORF52 as the viral protein underlying these changes. Remarkably, incoming virions were able to supply sufficient ORF52 to induce MT thickening and acetylation near the MTOC, potentially aiding in the delivery viral genomes to the nucleus. Although the function of MT alterations during late stages of infection requires further study, ORF52 shares functional and structural similarities with alphaherpesvirus VP22, underscoring the evolutionary importance of MT cytoskeletal manipulations for this virus family., (Copyright © 2017 Loftus et al.)

HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context., Importance: Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system is composed of a translocation channel and an extracellular T pilus. Bitopic VirB10, the VirB7 lipoprotein, and VirB9 interact to form a cell envelope-spanning structural scaffold termed the "core complex" that is required for the assembly of both structures. The related pKM101-encoded core complex is composed of 14 copies each of these VirB homologs, and the transmembrane (TM) α helices of VirB10-like TraF form a 55-Å-diameter ring at the inner membrane. Here, we report that the VirB10 TM helix possesses two types of putative dimerization motifs, a GxxxA (GA4) motif and two leucine (Leu1, Leu2) zippers. Mutations in the Leu1 motif disrupted T-pilus biogenesis, but these or other mutations in the GA4 or Leu2 motif did not abolish substrate transfer. Replacement of the VirB10 TM domain with a nondimerizing poly-Leu/Ala TM domain sequence also blocked pilus production but not substrate transfer or formation of immunoprecipitable complexes with the core subunits VirB7 and VirB9 and the substrate receptor VirD4. The VirB10 TM helix formed weak homodimers in Escherichia coli, as determined with the TOXCAT assay, whereas replacement of the VirB10 TM helix with the strongly dimerizing TM helix from glycophorin A blocked T-pilus biogenesis in A. tumefaciens. Our findings support a model in which VirB10's TM helix contributes to the assembly or activity of the translocation channel as a weakly self-interacting membrane anchor but establishes a heteromeric TM-TM helix interaction via its Leu1 motif that is critical for T-pilus biogenesis.

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Delta 198-295), D-6 (Delta 322-416), and D-LZ (Delta 409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.

The K8 gene of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) shares many functional similarities with the BZLF1 gene of Epstein-Barr virus. The protein products of K8 and BZLF1, K-bZIP (RAP, K8) and Zta (BZLF1, ZEBRA, Z) have both been proposed to be members of the bZIP family of transcription factors, forming multimers via a coiled-coil motif termed a leucine zipper. Substantial evidence supporting this model for Zta is published. Here, we demonstrate that the proposed leucine zipper region of K-bZIP (amino acids 182 to 218) is required for multimer formation but that it does not fold as a coiled coil.

Epstein-Barr virus nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization and functions as a regulator of viral and cellular transcription. EBNA 3C contains glutamine-rich and proline-rich domains and a region in the N terminus consisting of a stretch of basic residues followed by a run of leucine residues spaced seven amino acids apart. This N-terminal domain is widely believed to represent a leucine zipper dimerization motif (bZIP). We have performed the first structural and functional analysis of this motif and demonstrated that this domain is not capable of forming stable homodimers. Peptides encompassing the EBNA 3C zipper domain are approximately 54 to 67% alpha-helical in solution but cannot form dimers at physiologically relevant concentrations. Moreover, the EBNA 3C leucine zipper cannot functionally substitute for another homodimerizing zipper domain in domain-swapping experiments. Our data indicate, however, that the EBNA 3C zipper domain behaves as an atypical bZIP domain and is capable of self-associating to form higher-order alpha-helical oligomers. Using directed mutagenesis, we also identified a new role for the bZIP domain in maintaining the interaction between EBNA 3C and RBP-Jkappa in vivo. Disruption of the helical nature of the zipper domain by the introduction of proline residues reduces the ability of EBNA 3C to inhibit EBNA 2 activation and interact with RBP-Jkappa in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.

DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E. coli and Agrobacterium tumefaciens. DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E. coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP. These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E. coli and A. tumefaciens. Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites. The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments. DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A. tumefaciens T-DNA transfer system, in E. coli and homotypic complexes of VirB10 in A. tumefaciens. VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives. Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.

The murine coronavirus spike (S) protein contains a leucine zipper domain which is highly conserved among coronaviruses. To assess the role of this leucine zipper domain in S-induced cell-to-cell fusion, the six heptadic leucine and isoleucine residues were replaced with alanine by site-directed mutagenesis. The mutant S proteins were analyzed for cell-to-cell membrane fusion activity as well as for progress through the glycoprotein maturation process, including intracellular glycosylation, oligomerization, and cell surface expression. Single-alanine-substitution mutations had minimal, if any, effects on S-induced cell-to-cell fusion. Significant reduction in fusion activity was observed, however, when two of the four middle heptadic leucine or isoleucine residues were replaced with alanine. Double alanine substitutions that involved either of the two end heptadic leucine residues did not significantly affect fusion. All double-substitution mutant S proteins displayed levels of endoglycosidase H resistance and cell surface expression similar to those of the wild-type S. However, fusion-defective double-alanine-substitution mutants exhibited defects in S oligomerization. These results indicate that the leucine zipper domain plays a role in S-induced cell-to-cell fusion and that the ability of S to induce fusion may be dependent on the oligomeric structure of S.

Neutralization resistance of human immunodeficiency virus type 1 (HIV-1) is a major impediment to vaccine development. We have found that residues of HIV-1 MN strain in the C terminus of gp120 and the leucine zipper (LZ) region of gp41 viral envelope proteins interact cooperatively to determine neutralization resistance and modulate infectivity. Further, results demonstrate that this interaction, by which regions of gp120 are assembled onto the LZ, involves amino acid residues intimately related to those which participate in the binding of the envelope to its receptor and coreceptor. Variations in this critical assembly structure determine the concordant, interdependent evolution of increased infectivity efficiency and neutralization resistance phenotypes of the envelopes. The results elucidate important structure-function relationships among epitopes that are important targets of vaccine development.

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615-3619, 1993; S. S.-L. Chen, J. Virol. 68:2002-2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation beta-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associated gag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.

Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and induces T lymphomas in chickens within weeks after infection. Only a limited number of viral transcripts are detected in MDV tumor samples and cell lines. One of the major transcripts encodes MEQ, a 339-amino-acid bZIP protein which is homologous to the Jun/Fos family of transcription factors. The C-terminal half of MEQ contains proline-rich repeats and, when fused to the DNA-binding domain of a yeast transcription factor, Gal4 (residues 1 to 147), exhibits transactivation function. MEQ can dimerize with itself and with c-Jun. The MEQ-c-Jun heterodimers bind to an AP-1-like enhancer within the MEQ promoter region with greater affinity than do homodimers of either protein, and they transactivate MEQ expression. Here we show that MEQ is expressed in the nucleus but, interestingly, with a predominant fraction in the nucleoli and coiled bodies. This makes MEQ the first bZIP protein to be identified in the nucleoli. MEQ contains two stretches of basic residues, designated basic region 1 (BR1) and basic region 2 (BR2). Using a series of deletion mutants, we have mapped the primary nuclear localization signal (NLS) and the sole nucleolar localization signal (NoLS) to the BR2 region. BR1 was shown to provide an auxiliary signal in nuclear translocation. To demonstrate that BR2 is an authentic NoLS, BR2 was fused to cytoplasmic v-Raf (delta gag) kinase. The BR2-Raf fusion protein was observed to migrate into the nucleoplasm and the nucleolus. The BR2 region can be further divided into two long arginine-lysine stretches, BR2N and BR2C, which are separated by the five amino acids Asn-Arg-Asp-Ala-Ala (NRDAA). We provide evidence that the requirement for nuclear translocation is less stringent than that for nucleolar translocation, as either BR2N or BR2C alone is sufficient to translocate the cytoplasmic v-Raf (delta gag) into the nucleus, but only in combination can they translocate v-Raf (delta gag) into the nucleolus. Our studies demonstrate that MEQ is both a nuclear and nucleolar protein, adding MEQ to the growing list of transactivators which localize to the nucleolus.

The nuclear protein v-Myb, encoded by the avian myeloblastosis virus (AMV), can induce acute monoblastic leukemia in vivo and transform chicken myelomonocytic cells in culture. The N terminus of v-Myb functions as the DNA-binding domain, and multiple central and C-terminal regions of this protein have been reported to function in transcriptional activation of model reporter genes. We showed previously that a C-terminal domain (amino acids 296 to 371) is required for transcriptional activation and transformation of primary chicken myelomonocytic cells. In this study, we have now analyzed a series of C-terminal mutants of v-Myb to further investigate this domain. A strong correlation was observed between transcriptional activation and leukemic transformation by this series of mutants. Furthermore, deletion analyses demonstrate that the C-terminal 41 amino acids of v=MybAMV (amino acids 331 to 371 of the Myb portion) are nonessential whereas further deletion of amino acids 321 to 330 (EFAETLQLID) results in a nonfunctional protein. Hence, we defined a 10-amino-acid subregion (the "FAETL" motif) required for transcriptional activation and oncogenic transformation by v-Myb Amv. The FAETL region is part of a putative leucine zipper structure and lies near a cluster of phosphorylation sites. Our analysis of mutants with substitutions of the zipper leucines or multiple adjacent phosphorylation sites demonstrates that the function of the FAETL motif is not dependent on an intact leucine zipper structure or adjacent phosphorylation sites. The study of GAL4-Myb fusions suggests that this region is important in maintaining a fully functional conformation of v-Myb. The putative leucine zipper structure has previously been proposed to exert inhibitory effects on c-Myb because its mutation caused increased transcriptional transactivation and transformation. Interestingly, our results show that this region is essential for the functions of v-Myb without requiring a heptad leucine repeat.

We have previously reported that synthetic peptides representing the leucine zipper domain (DP107) and a second putative helical domain (DP178) of human immunodeficiency virus type 1 (HIV-1) gp41 exhibit potent anti-HIV activity. In this study we have used soluble recombinant forms of gp41 to provide evidence that the DP178 peptide and the DP178 region of gp41 associate with a distal site on the gp41 transmembrane protein whose interactive structure is influenced by the leucine zipper (DP107) motif. We also observed that a single coiled-coil-disrupting mutation in the leucine zipper domain transformed the recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion and infectivity, which may be related to that finding. We speculate that this transformation results from liberation of the potent DP178-related sequence from a molecular clasp with a leucine zipper, DP107, determinant. The results are discussed in the context of two distinct conformations for the gp41 molecule and possible involvement of these two domains in structural transitions associated with HIV-1-mediated fusion. The results are also interpreted to suggest that the anti-HIV activity of the various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic processes.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.

Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines.

The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein, represses the MIEP through a DNA sequence located between the TATA box and the transcription initiation site, designated the cis repression signal (crs). Through mutational analysis, amino acid domains within IE86 responsible for binding the crs element were located at the C terminus. Mutation of the putative zinc finger domain, which precluded IE86 from binding DNA, converted the protein from a repressor of MIEP transcription into an activator. DNase I protection analysis demonstrated that the IE86 footprint overlapped the sequence protected by the TATA-binding protein (TBP). Investigation of whether IE86 was able to displace TBP from DNA revealed that both proteins could bind DNA simultaneously. However, higher concentrations of IE86 were required to obtain protection of the crs element in the presence of prebound TBP. Similarly, higher concentrations of TBP were required to obtain protection in the presence of prebound IE86. These observations indicate that steric hinderance impairs but does not prevent both proteins from binding DNA synchronously.

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.

The Epstein-Barr virus BZLF1 gene product EB1 (also called ZEBRA and Zta), is a transcription factor belonging to the bZIP (basic domain leucine zipper) family of nuclear proteins. Translocation to the nucleus of EB1 (J. Becker, U. Leser, M. Marschall, A. Langford, W. Jilg, H. Gelderblom, P. Reichart, and H. Wolf, Proc. Natl. Acad. Sci. USA 88:8332-8336, 1991) and of two other bZIP proteins, c-Jun and c-Fos (P. Roux, J.-M. Blanchard, A. Fernandez, N. Lamb, P. Jeanteur, and M. Piechaczyk, Cell 63:341-351, 1990), has been shown to be subject to regulation. We show here that for both EB1 and Jun the nuclear targeting signals (NTS) in the proteins' primary sequences are two clusters of positively charged amino acids. These clusters, called BRA and BRB, are necessary and sufficient to direct beta-galactosidase to the nuclear compartment and act as a bipartite NTS. They are conserved among all the bZIP proteins, and although they are not identical, they probably share the same function. Site-directed mutagenesis studies made on these basic clusters suggest that they also act as a bipartite NTS in the EB1 protein. Our results also demonstrate that in EB1 and Jun, these bipartite NTS are superimposed with bipartite DNA-binding domains, since BRA and BRB are required in vitro for direct and specific contact between these proteins and their DNA-binding sites.

Using peptide antisera specific for regions within the N terminus and C terminus of the predicted UL36 gene product, immunoblotting experiments were performed to demonstrate definitively that ICP1/2 is encoded by the UL36 gene. These data also suggest that both the cell- and the virion-associated forms of ICP1/2 are colinear with the complete predicted amino acid sequence of the UL36 gene. Computer-assisted analyses of the predicted amino acid sequence of the UL36 gene revealed the presence of two putative leucine zipper-type motifs and a potential ATP-binding domain. The possible functions of these consensus domains will also be discussed.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.

Many retroviruses, including the human and simian immunodeficiency viruses, contain a leucine zipper-like repeat in a highly conserved region of the external domain of the transmembrane (TM) glycoprotein. This region has been postulated to play a role in stabilizing the oligomeric form of these molecules. To determine what role this region might play in envelope structure and function, several mutations were engineered into the middle isoleucine of the leucine zipper-like repeat of the human immunodeficiency virus type 1 (HIV-1) TM protein. A phenotypic analysis of these mutants demonstrated that conservative mutations (Ile to Val or Leu) did not block the ability of the viral glycoprotein to mediate cell-cell fusion or affect virus infectivity. In contrast, each of the other mutations, except for the Ile-to-Ala change, completely inhibited the ability of the glycoprotein to fuse HeLa-T4 cells and of mutant virions to infect H9 cells. The alanine mutation produced an intermediate phenotype in which both cell fusion and infectivity were significantly reduced. Thus, the biological activity of the glycoprotein titrates with the hydrophobicity of the residue in this position. None of the mutations affected the synthesis, oligomer formation, transport, or processing of the HIV glycoprotein complex. Although these results do not rule out a role for the leucine zipper region in glycoprotein oligomerization, they clearly point to a critical role for it in a post-CD4 binding step in HIV membrane fusion and virus entry.

In Escherichia coli, a yopE::lacZ fusion was found to be regulated by temperature in the presence of the cloned BamHI G fragment of Yersinia pestis plasmid pCD1, which contains the lcrF locus. Increasing the copy number of lcrF relative to that of the yopE reporter had a negligible effect on the induction ratio (26 versus 37 degrees C) but caused large reductions in the absolute levels of yopE transcription. We localized the lcrF gene by monitoring the induction phenotype of BamHI G deletion derivatives. Sequencing revealed an open reading frame capable of encoding a protein of 30.8 kDa. A protein product of this size was detected in a T7 expression system, and LcrF-dependent yopE-specific DNA binding activity was observed. As expected, LcrF exhibited 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcriptional regulatory proteins. These proteins could be divided into two classes according to function: those regulating operons involved in catabolism of carbon and energy sources and those involved in regulating virulence genes. lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. A portion of LcrF was found associated with the membrane fraction in E. coli; however, pulse-chase experiments indicated that this result is an artifact of fractionation.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.

The UL9 gene of herpes simplex virus (HSV) codes for a DNA-binding protein (OBP) that interacts sequence specifically with the origin of replication. This protein is essential for HSV DNA replication in cultured cells. The UL9 gene was cloned into a plasmid vector downstream of the SP6 RNA polymerase promoter. By using in vitro transcription and translation systems, a full-length OBP was synthesized. This synthetic protein is recognized by an antiserum generated against the C-terminal decapeptide of OBP and is functionally active in binding to OriS sequence specifically. The in vitro-synthesized protein has sequence specificity for binding similar to that found for the in vivo-generated OBP. A total of 14 in-frame deletion and insertion mutants of the UL9 gene were generated and expressed in vitro. Using these deletion mutants, we determined that the 269-amino-acid stretch defined by amino acids 564 to 832 localizes the OriS-specific DNA-binding domain. The N-terminal boundary is between amino acids 565 and 596, while the C terminus lies between amino acids 833 and 805. This segment contains a helix-turn-helix moiety and a pseudo-leucine zipper, neither of which alone can support DNA binding. The other leucine zipper from amino acids 150 to 173 is not required for the in vitro sequence-specific DNA-binding activity of OBP.