Sucrose Gradient Sedimentation Research Articles

Mutant Huntingtin Blocks BDNF Transport

The mechanisms by which polyglutamine (polyQ) expansion in the huntingtin protein (htt) cause neuronal pathogenesis are unclear (see Ross). Gauthier et al. report that wild-type huntingtin is required for proper vesicular transport of brain-derived neurotrophic factor (BDNF) through its interactions with huntingtin-associated protein-1 (HAP1), which in turn interacts with the p150 Glued subunit of dynactin, a required regulator of the microtubule motor dynein. Vesicle movement was monitored by recording the movement of BDNF-eGFP (enhanced green fluorescent protein)-tagged vesicles by ultrafast 3D videomicroscopy in NG108-15 neuroblastoma cells expressing either wild-type htt or polyQ-htt. Only wild-type htt promoted the movement of vesicles, measured as either the proportion of moving vesicles or the velocity of vesicle movement. To determine the relevance to Huntington's disease, the authors analyzed BDNF vesicular transport rates in wild-type cells, cells with one mutant htt allele (polyQ-htt), and cells with two mutant htt alleles. Cells with polyQ-htt showed decreased vesicle velocity, and overexpression of wild-type htt increased the velocity of vesicle movement. HAP1 was required for the htt stimulation of vesicle movement, because reduction of HAP1 by small interfering RNA blocked htt's stimulation of transport, and htt truncation mutants that did not interact with HAP1 did not stimulate transport. Sucrose gradient sedimentation analysis of wild-type cells or polyQ-htt cells indicated that htt was present in the dynein and dynactin protein complex, whereas polyQ-htt was present in a lower density fraction. In cells from Huntington patients, a disruption in the dynein and dynactin complex was observed. PolyQ-htt inhibited the interaction of HAP1 and various motor proteins (components of both kinesin and dynein) with microtubules. Release of BDNF after an initial stimulation was compromised in cells with polyQ-htt. Thus, the disruption of vesicular transport by polyQ-htt appears to block the refilling of the releasable pool of BDNF, which results in loss of trophic support and cell death. C. A. Ross, Huntington's disease: New paths to pathogenesis. Cell 118 , 4-7 (2004). [Online Journal] L. R. Gauthier, B. C. Charrin, M. Borrell-Pagès, J. P. Dompierre, H. Rangone, F. P. Cordelières, J. de Mey, M. E. MacDonald, V. Leßmann, S. Humbert, F. Saudou, Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 118 , 127-138 (2004). [Online Journal]

Mitogenic activity of Epstein-Barr virus-encoded BARF1 protein.

We previously reported that BARF1 gene has either an immortalizing effect, when expressed in primary primate epithelial cells, or a malignant transforming activity, when expressed in established and nontumoral rodent fibroblast or human B-cell lines. As predicted from sequence analysis, we found that BARF1 coded protein can be secreted from different cell lines, among them BARF1-transfected Balb/c3T3 rodent fibroblasts. Thus, as an initial step to clarify BARF1 oncogenic functions, we investigated whether the secreted form of BARF1 protein can activate the cell cycle as a growth factor. Since efficient BARF1 expression could be obtained from 293-tTA cells infected with a tetracycline-regulatable recombinant adenovirus, secreted BARF1 product could be purified from the culture medium of such cells by ammonium sulfate precipitation, ion exchange chromotography and sucrose gradient sedimentation. We describe in this paper that addition of a purified product of secreted BARF1 protein to serum-free culture medium of Balb/c3T3 rodent fibroblasts, human Louckes B-cell line and primary monkey kidney epithelial cells resulted in a cell cycle activation that was inhibited by affinity-purified anti-BARF1 antibody. Our demonstration of a specific stimulation of cell cycle in vitro by BARF1 secreted product suggests that this EBV-encoded BARF1 protein could act as a growth factor in vivo.

Translational upregulation of yes accompanies eIF4E-mediated oncogenic transformation

Overexpression of the translation initiation factor eIF4E results in transformation of normal fibroblasts as a single-hit oncogene. This implies that eIF4E must affect several pathways leading to transformation. The oncogenic potential of eIF4E is probably realized by elevating the translational efficiency of some oncogene and growth-promotion transcripts that are normally repressed by their 5'UTR (untranslated region). To address this possibility, we have cloned mRNAs whose polysomal representation increases upon overexpression of eIF4E. Among these mRNAs, we now report the isolation of a clone corresponding to the src-like kinase yes. The yes mRNA contains a long 5'UTR with characteristic features of a typical translationally repressed transcript. This was confirmed by analysis of the distribution of yes mRNA after sedimentation in sucrose gradients. Increased utilization of yes mRNA resulted in elevated expression of the protein product in cells transformed with eIF4E, and suggested that overexpression of Yes could contribute to eIF4E-mediated transformation. To test this, we monitored the malignant properties of MM3MG-4E cells after treatment with PP2, a specific inhibitor of src kinases. Growth in soft agar and saturation densities were significantly reduced after treatment with PP2, but treatment of mice harboring MM3MG-4E tumors with PP2 did not affect tumor growth. However, transformation of yes-null fibroblasts by eIF4E was significantly impaired.

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts

Cardiac ATP-sensitive K + (K ATP) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K ATP channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2–SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2) 4–(SUR2A) 4, suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K ATP channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.

Analysis of Estrogen Receptor Expression in Tissues

This chapter focuses on the analysis of estrogen receptor expression in tissues. The functions of estrogen are not confined to the female reproductive system, but it also has important functions in non reproductive tissues of both males and females. The two estrogen receptors—ERα and ERβ—have distinct and sometimes opposing actions in multiple tissues. To define the role of each receptor in estrogen-sensitive tissues, it is essential to have reliable methods to detect the expression of the two genes. Methods have been developed for the detection of ERα and its functions. The most common of these are immunohistochemistry and Western blotting with specific antibodies; the measurement of estrogen accumulation and retention in tissues; the measurement of the binding of estradiol in the 8S fraction after sedimentation in sucrose gradients of low salt tissue-extracts; and the assessment of changes in mRNA levels of classic estrogen responsive genes. There are several reasons why the presence of the second estrogen receptor has made it difficult to interpret results obtained with the use of these methods. Available estrogen receptor antibodies when tested with recombinant ERα or ERβ standards exhibit the appropriate specificity; however, this recognition does not indicate them to be high affinity antibodies.

Characterization of Ad5 E3–14.7K, an adenoviral inhibitor of apoptosis: Structure, oligomeric state, and metal binding

The adenovirus E3-14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF-alpha and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3-14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five beta-strands and one alpha-helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution.

Erythroid Expression and Oligomeric State of the AQP3 Protein

Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.

Internalization of echovirus 1 in caveolae.

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

Conserved region CR2 of Epstein-Barr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association.

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate gamma-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401-413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

Mechanisms of dCMP Transferase Reactions Catalyzed by Mouse Rev1 Protein

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Overexpression of the adenovirus type 12 (Ad12) pTP or E1A gene facilitates Ad12 DNA replication in nonpermissive BHK21 hamster cells.

In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.

A DnaJ-like protein homologous to the yeast co-chaperone Sis1 (TcJ6p) is involved in initiation of translation in Trypanosoma cruzi.

In eukaryotes, proteins homologous to the bacterial DnaJ protein are involved in regulation of the Hsp70 molecular chaperones, which are implicated in a variety of protein biogenesis pathways. We report herewith the molecular characterization of a T. cruzi DnaJ gene, termed TcJ6, encoding a protein that displays high sequence homology with the Saccharomyces cerevisiae Sis1 co-chaperone required for the initiation of translation. TcJ6 protein was expressed as a polypeptide of 36.5 kDa at a constant level during parasite differentiation and was associated to the cytoplasmic fraction. We showed that overexpression of TcJ6 complemented a temperature-sensitive yeast sis1 mutant. In addition, sucrose gradient sedimentation analysis of polysomes from T. cruzi and a yeast mutant overexpressing TcJ6p showed that the trypanosomal co-chaperone was closely associated with ribosomal subunits, 80 S monosomes and the smaller polysomes, as observed for Sis1p. Furthermore, in T. cruzi TcJ6p was also found to be preferentially concentrated around the nucleus, giving a speckled staining pattern. This suggests that TcJ6p is associated with the endoplasmic reticulum. Taken together, these data suggest that the trypanosomal DnaJ is involved in initiation of translation.

VSV-G envelope glycoprotein forms complexes with plasmid DNA and MLV retrovirus-like particles in cell-free conditions and enhances DNA transfection.

We have previously shown that vesicles containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) can associate efficiently with immature, non-infectious, envelope-deficient retrovirus-like particles assembled by packaging cells to produce infectious, pseudotyped viruses in cell-free conditions in vitro. We have also previously reported that VSV-G can enhance DNA lipofection efficiency by interacting with liposomes to form fusogenic, serum-stable liposomes with enhanced transfection properties. Here, we report that VSV-G can form a complex directly with naked plasmid DNA in the absence of a lipofection reagent and can thereby enhance the transfection efficiency of the naked plasmid vector. Sucrose gradient sedimentation analysis demonstrated that VSV-G can also associate with plasmid DNA and murine leukemia virus (MLV) gag-pol particles to form ternary complexes that co-sediment with high DNA transfecting activity. The increased transfection efficiency with VSV-G was dependent on the presence of the polycation (Polybrene) in the culture medium during transfection. Enhanced transfection was abolished by a neutralizing antibody to VSV-G. These results may be useful in the study of retrovirus assembly, in the further design of hybrid DNA-based retrovirus-like vectors, and in the full in vitro, cell-free assembly of infectious virus-like particles from component parts.

Characterization of the Mammalian Peroxisomal Import Machinery: Pex2p, Pex5p, Pex12p, AND Pex14p ARE SUBUNITS OF THE SAME PROTEIN ASSEMBLY

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.

Carotenoid binding to photosystems I and IIof Chlamydomonas reinhardtii cells grown under weak light or exposed to intense light

Thylakoids, isolated from Chlamydomonas reinhardtii cells which were grown under weak light or exposed to a short period of intense light, were solubilised with decanoyl-N-methylglucamide (Mega 10) or with a new related detergent, dodecanoyl-N-hydroxyethylglucamide (DODHEGA) to prepare photosystem I and II complexes or sub-complexes for pigment stoichiometry determination. Loroxanthin was only bound to photosystem II antenna but most of the lutein, the other main α-carotene derived xanthophyll, was also associated with photosystem II antenna, as well as with the photosystem I antenna in which it represented the main xanthophyll species. The mean xanthophyll composition of the photosystem II antenna, prepared both by partially denaturing electrophoresis or sucrose gradient sedimentation, showed that more than three molecules (most probably four) were associated with individual light-harvesting complex II (LHCII) polypeptide (on the basis of seven chl a per polypeptide). Intense light exposition of the cells resulted in increased levels of lutein and of components of the violaxanthin cycle, part of them being only loosely bound to pigment protein complexes. The violaxanthin cycle components were recovered in PSI and PSII whereas the additional level of lutein was restricted to PSII sub-complexes.

Identification and Characterization of a SET/NAP Protein Encoded by a Brain-Specific Gene, MB20

A new member of the NAP/SET gene family, named MB20, was isolated from a mouse brain cDNA library by virtue of its CAG trinucleotide repetitive sequence and a brain-specific gene expression pattern. The complementary DNA sequence predicted an open reading frame of 545 amino acids, with four copies of an 11-amino-acid direct repeat. The consensus sequence for these repeats, PKE-P--K-EE, is present in the largest subunit of murine neurofilament (NF-H). The MB20 protein sequence is homologous to nucleosome assembly proteins of several species, and its C-terminus is homologous to SET proteins. Immunoblot analysis revealed that MB20 protein is expressed in the brain. Transient transfection and immunofluorescence microscopy demonstrated that MB20 is distributed in the cytoplasm as well as in the nucleus. Deletion of the N-terminal end imparts the complete localization of MB20 protein to the nucleus. The ability of MB20 to bind histone proteins was analyzed by sucrose gradient sedimentation and by retention of histone proteins by immobilized MB20 protein. On the basis of its expression pattern, predicted sequence, and protein properties, we propose that MB20 plays a unique role in modulating nucleosome structure and gene expression during brain development.

Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His 6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His 6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

The Fate of Membrane-bound Ribosomes Following the Termination of Protein Synthesis

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.

Intracellular Cre-Mediated Deletion of the Unique Packaging Signal Carried by a Herpes Simplex Virus Type 1 Recombinant and Its Relationship to the Cleavage-Packaging Process

To gain further insight on the function of the herpes simplex virus type 1 (HSV-1) packaging signal (a sequence), we constructed a recombinant virus containing a unique a sequence, which was flanked by two loxP sites in parallel orientation. The phenotype of this recombinant, named HSV-1 LaL, was studied in cell lines which either express or do not express Cre recombinase. Although LaL virus multiplication was only slightly reduced in standard cell lines, its growth was strongly inhibited in Cre-expressing cells. In these cells, a sequences were detected mostly in low-molecular-weight DNA circles, indicating that they had been excised from virus DNA by site-specific recombination. Deletion of the a sequences from the viral genome resulted in the accumulation of uncleaved replication intermediates, as observed by pulsed-field gel electrophoresis. B-type capsids also accumulated in these cells, as shown both by electron microscopy and by sucrose gradient sedimentation. Further examination of the status of a sequences in Cre-expressing cells indicated that high-level amplification of this sequence can occur in the absence of the cleavage-packaging process. Moreover, the amplified a signals in small circular DNA molecules remained uncleaved, indicating that these molecules were not able to efficiently interact with the cleavage-packaging machinery. The cleavage-packaging machinery and the structural proteins required to assemble virions were, however, functional in HSV-1 LaL-infected Cre-expressing cells, since this system could be used to package plasmid DNA harboring an origin of virus replication and one normal a signal. This is the first study in which accumulation both of uncleaved replication intermediates and of B capsids has been obtained in the presence of the full set of proteins required to package virus DNA.