Heterogeneous Functional Ty1 Elements Are Abundant in the Saccharomyces Cerevisiae Genome

Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(Δ475-3944)HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.     

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.     

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH₂-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH₂ or the COOH termini of heterologous proteins.

We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

     

Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication,and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Δ⁴⁹¹RSRR⁴⁹⁴ and point mutation ⁴⁹¹GSGG⁴⁹⁴ in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Δ⁴⁹¹RSRR⁴⁹⁴ or ⁴⁹¹GSGG⁴⁹⁴ mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ or pOka-gB⁴⁹¹GSGG⁴⁹⁴ virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16). Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12, 27, 57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10, 19, 38, 48, 51, 52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N- and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homology-like domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long α helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming the core of the trimer making contacts with the two other subunits. The structural homology and lack of furin cleavage suggest that the herpesvirus gB and VSV G proteins have undergone convergent evolution.Although not proven experimentally, VZV gB is likely to be cleaved by the subtilisin-like proprotein convertase furin as the glycoprotein has a furin recognition motif [R]-X-[KR]-R-|-X (where | indicates the position at which the polypeptide is cleaved) (29). The [R]-X-[KR]-R-|-X motif is conserved in gBs for all of the herpesvirus families (5, 9, 21, 36, 40, 53, 63, 64). This site has been shown to be dispensable for the replication of human cytomegalovirus (HCMV), bovine herpesvirus type 1 (BHV-1), and pseudorabies virus (PRV) in vitro (32, 49, 58). Furin site mutants for BHV-1 and PRV show an altered phenotype in vitro, but effects were not examined in vivo. HSV-1 gB is not cleaved and lacks the [R]-X-[KR]-R-|-X motif at the canonical site, which is of interest because HSV-1 is genetically the most closely related human herpesvirus to VZV.Domain I of HSV gB showed structural conservation of putative fusion loops similar to those found in domain IV of the VSV G protein (28). Despite the lack of conserved amino acids within these loops, the hydrophobicity of the residues appears to be conserved for the Herpesviridae (4). Substitution of hydrophobic residues in Epstein-Barr virus gB and linker insertion mutagenesis close to the putative fusion loops of HSV-1 gB abrogated fusion based on in vitro transfection studies (4, 22, 34). However, the effect of substitutions in these putative fusion loops on viral replication has not been characterized. Since the development of fusion assays for VZV has proven elusive, the effect of substitutions in the putative fusion loop using viral mutagenesis to make recombinant viruses provides an alternative approach for identifying functional residues in VZV gB.In contrast to HSV-1, VZV is a human-restricted pathogen (reviewed in reference 16). To study the pathogenesis of VZV in vivo, well-established human xenograft models have been developed using SCID mice (6, 7, 13, 14, 41, 44, 54, 65). Lesions formed by VZV in the skin are similar to those seen in human subjects following primary infection (15, 43). The relevance of the model was demonstrated by studies with the varicella vaccine virus (vOka) that exhibited decreased growth in skin xenografts in vivo but does not cause disease in the healthy human host. In contrast, the vaccine virus and its parent strain, parental Oka (pOka), have indistinguishable replication kinetics in vitro (15, 43).The present study was designed to investigate the effects of structure-based targeted mutations in VZV gB on viral replication in cultured cells and in human skin xenografts in the SCIDhu mouse model. This was performed in the context of infectious virus recovered using the self-excisable bacterial artificial chromosome (BAC) containing the genome of a clinical isolate, Oka (62). The roles of the conserved residues W180 (gB-W180G) and Y185 (gB-Y185G) in the putative fusion loop were evaluated using glycine substitution, and the role of the furin recognition motif (⁴⁹¹RSRR⁴⁹⁴) was assessed by a complete deletion of the furin motif (gBΔ⁴⁹¹RSRR⁴⁹⁴) or a substitution of the arginine residues with glycine (gB⁴⁹¹GSGG⁴⁹⁴) to conserve the carbon backbone.     

In Vivo Analysis of the Notch Receptor S1 Cleavage

A ligand-independent cleavage (S1) in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.     

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34⁺ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.     

Protease Cleavage Sites in HIV-1 gp120 Recognized by Antigen Processing Enzymes Are Conserved and Located at Receptor Binding Sites

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.A major goal of HIV vaccine development is the development of immunogens that elicit protective antiviral antibody and cellular immune responses. However, after more than 25 years of research, vaccine immunogens able to elicit protective immunity in humans have yet to be described (11, 31). Although it has been possible to produce recombinant envelope proteins (gp120 and gp140) with many of the features of native virus proteins (e.g., complex glycosylation and the ability to bind CD4, chemokine receptors, and neutralizing antibodies), these antigens have not been able to elicit broadly neutralizing antibodies (bNAbs) or protective immune responses when used as immunogens (11, 32, 43, 50, 56, 74, 79). The fact that recombinant proteins can adsorb virus bNAbs from HIV-1-positive sera (59, 91) indicates that many recombinant envelope proteins are correctly folded but that the epitopes recognized by bNAbs are simply not immunogenic. Over the last decade, several different approaches have been employed to create immunogens able to elicit broadly neutralizing antibodies. These strategies have included efforts to duplicate and/or stabilize the oligomeric structure of HIV envelope proteins (5, 26, 87), the creation of minimal antigenic structures lacking epitopes that conceal important neutralizing sites (27, 46, 70, 89), and prime/boost strategies combining protein immunization with DNA immunization or infection with recombinant viruses in order to stimulate the endogenous synthesis and presentation of HIV immunogens (15, 29, 30, 83). However, none of these approaches has resulted in a clinically significant improvement in antiviral immunity or HIV vaccine efficacy. Efforts to elicit protective cellular immune responses (e.g., cytotoxic lymphocytes) by use of recombinant virus vaccines have likewise been disappointing (10, 61). In fact, such vaccines may have promoted HIV infection rather than inhibiting it (22, 23).In the present study, we describe the first steps in a new approach to reengineering the immunogenicity of HIV envelope proteins in order to improve the potency and specificity of humoral and cellular immune responses. The approach is based on defining the determinants of antigen processing and presentation of HIV envelope glycoproteins. Both humoral and cellular immune responses depend on proteolytic degradation of protein antigens prior to antigen presentation, mediated by professional antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells (97). Normally, proteins of intracellular origin are processed by the proteasome, a 14- to 17-subunit protein complex located in the cytosol. Proteins of extracellular origin are processed in lysosomes or late endosomes of APCs. The resulting peptide epitopes are then loaded into major histocompatibility complex (MHC) class I or class II molecules and presented on the surfaces of APCs to CD8 or CD4 T cells. Within the endosomes and lysosomes of APCs, there are cathepsins, acid thiol reductase, and aspartyl endopeptidase. The enzymes perform two activities: degrading endocytosed protein antigens to liberate peptides for MHC class II binding (99) and removing the invariant chain chaperone (6, 94). Although all cathepsins can liberate epitopes from a diverse range of antigens (16), only cathepsins S and L have nonredundant roles in antigen processing in vivo (reviewed by Hsing and Rudensky [45]). Cathepsin L is expressed in thymic cortical epithelial cells but not in B cells or dendritic cells, while cathepsin S is found in all three types of APCs. Unlike cathepsins L and S, which are cysteine proteases and active at neutral pH, cathepsin D is an aspartic protease, is active at acidic pH, and participates in proteolysis and antigen presentation in connection with MHC class I and class II antigen presentation pathways established for CD4 and CD8 T cells. In considering the use of envelope proteins as potential vaccines, the route of immunization, formulation (e.g., adjuvants), protein folding, disulfide bonding, and glycosylation pattern all determine which peptides are available for MHC-restricted presentation.Previous studies provided evidence that gp120 was sensitive to digestion by cathepsins B, D, and L, but the specific cleavage sites were not defined (18). In the present study, we (i) describe the locations of eight protease cleavage sites on HIV-1 gp120 recognized by cathepsins L, S, and D, involved in antigen processing; (ii) determine the extent to which they are conserved; and (iii) evaluate the effect of cathepsin cleavage on the binding of gp120 to CD4-IgG and neutralizing antibodies. The results obtained provide new insights into the basis of envelope immunogenicity that may prove to be useful in the development of HIV vaccine antigens.     

Genes for Polymorphic H1 Histones Are Linked in the Japanese Quail Genome

In a previous report (Palyga, J., Biochem.Genet. 29, 431-445, 1991), three subtypes oferythrocyte histone H1 were found to vary in a Japanesequail population. While H1.b and H1.z histones were eachrepresented by two electromorphs differing in apparentmolecular weights, a polymorphism of histone H1.a wasconnected with a lack of this protein in some birds. Asa genetic basis for this variability was demonstrated only in H1.b, here genetic data are providedwhich indicate that both H1.a and H1.z are encoded bytwo codominant alleles at a locus. A linkage analysis offamily data in 13 quail pedigrees has revealed a significant linkage between H1.a and H1.z andbetween H1.b and H1.z ( lod scores about 12 and 5,respectively). Thus, a gene for histone H1.z is locatedbetween H1.a and H1.b in the quail genome.