HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

Circular dichroism in hard X-ray photoelectron diffraction observed by time-of-flight momentum microscopy.

X-ray photoelectron diffraction (XPD) is a powerful technique that yields detailed structural information of solids and thin films that complements electronic structure measurements. Among the strongholds of XPD we can identify dopant sites, track structural phase transitions, and perform holographic reconstruction. High-resolution imaging of kll-distributions (momentum microscopy) presents a new approach to core-level photoemission. It yields full-field kx-ky XPD patterns with unprecedented acquisition speed and richness in details. Here, we show that beyond the pure diffraction information, XPD patterns exhibit pronounced circular dichroism in the angular distribution (CDAD) with asymmetries up to 80%, alongside with rapid variations on a small kll-scale (0.1 Å-1). Measurements with circularly-polarized hard X-rays (hν = 6 keV) for a number of core levels, including Si, Ge, Mo and W, prove that core-level CDAD is a general phenomenon that is independent of atomic number. The fine structure in CDAD is more pronounced compared to the corresponding intensity patterns. Additionally, they obey the same symmetry rules as found for atomic and molecular species, and valence bands. The CD is antisymmetric with respect to the mirror planes of the crystal, whose signatures are sharp zero lines. Calculations using both the Bloch-wave approach and one-step photoemission reveal the origin of the fine structure that represents the signature of Kikuchi diffraction. To disentangle the roles of photoexcitation and diffraction, XPD has been implemented into the Munich SPRKKR package to unify the one-step model of photoemission and multiple scattering theory.

Optimal pacing strategy: from theoretical modelling to reality in 1500-m speed skating.

PURPOSE: Athletes are trained to choose the pace which is perceived to be correct during a specific effort, such as the 1500-m speed skating competition. The purpose of the present study was to "override" self-paced (SP) performance by instructing athletes to execute a theoretically optimal pacing profile. METHODS: Seven national-level speed-skaters performed a SP 1500-m which was analysed by obtaining velocity (every 100 m) and body position (every 200 m) with video to calculate total mechanical power output. Together with gross efficiency and aerobic kinetics, obtained in separate trials, data were used to calculate aerobic and anaerobic power output profiles. An energy flow model was applied to SP, simulating a range of pacing strategies, and a theoretically optimal pacing profile was imposed in a second race (IM). RESULTS: Final time for IM was ∼2 s slower than SP. Total power distribution per lap differed, with a higher power over the first 300 m for IM (637.0 (49.4) vs 612.5 (50.0) W). Anaerobic parameters did not differ. The faster first lap resulted in a higher aerodynamic drag coefficient and perhaps a less effective push-off. CONCLUSION: Experienced athletes have a well-developed performance template, and changing pacing strategy towards a theoretically optimal fast start protocol had negative consequences on speed-skating technique and did not result in better performance.

Suppression of the vacuum space-charge effect in fs-photoemission by a retarding electrostatic front lens.

The performance of time-resolved photoemission experiments at fs-pulsed photon sources is ultimately limited by the e-e Coulomb interaction, downgrading energy and momentum resolution. Here, we present an approach to effectively suppress space-charge artifacts in momentum microscopes and photoemission microscopes. A retarding electrostatic field generated by a special objective lens repels slow electrons, retaining the k-image of the fast photoelectrons. The suppression of space-charge effects scales with the ratio of the photoelectron velocities of fast and slow electrons. Fields in the range from -20 to -1100 V/mm for Ekin = 100 eV to 4 keV direct secondaries and pump-induced slow electrons back to the sample surface. Ray tracing simulations reveal that this happens within the first 40 to 3 µm above the sample surface for Ekin = 100 eV to 4 keV. An optimized front-lens design allows switching between the conventional accelerating and the new retarding mode. Time-resolved experiments at Ekin = 107 eV using fs extreme ultraviolet probe pulses from the free-electron laser FLASH reveal that the width of the Fermi edge increases by just 30 meV at an incident pump fluence of 22 mJ/cm2 (retarding field -21 V/mm). For an accelerating field of +2 kV/mm and a pump fluence of only 5 mJ/cm2, it increases by 0.5 eV (pump wavelength 1030 nm). At the given conditions, the suppression mode permits increasing the slow-electron yield by three to four orders of magnitude. The feasibility of the method at high energies is demonstrated without a pump beam at Ekin = 3830 eV using hard x rays from the storage ring PETRA III. The approach opens up a previously inaccessible regime of pump fluences for photoemission experiments.

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Mouse models for hereditary nonpolyposis colorectal cancer.

Hemizygous germ-line defects in mismatch repair (MMR) genes underlie hereditary nonpolyposis colorectal cancer (HNPCC). Loss of the wild-type allele results in a mutator phenotype, accelerating tumorigenesis. Tumorigenesis specifically occurs in the gastrointestinal and genitourinary tracts; the cause of this tissue specificity is elusive. To understand the etiology and tissue distribution of tumors in HNPCC, we have developed mouse models carrying a deficiency in the MMR gene Msh2. Most of the completely Msh2-deficient mice succumbed to lymphomas at an early age; lymphomagenesis was synergistically enhanced by exposure to ethylnitrosourea. Lymphomas were absent in immunocompromised Tap1-/-;Msh2-/- mice; these mice generally succumbed to HNPCC-like tumors. Together, these data suggest that the HNPCC tumor spectrum is determined by exposure of MMR-deficient cells to exogenous mutagens, rather than by tissue-specific loss of the wild-type MMR allele or by immune surveillance. Msh2 hemizygous mice had an elevated tumor incidence that, surprisingly, was rarely correlated with loss of the Msh2+ allele. To develop a model for intestinal tumorigenesis in HNPCC, we introduced the Min allele of the Apc tumor suppressor gene. We observed loss of the wild-type Msh2 allele in a significant fraction of intestinal tumors in Apc+/Min;Msh2+/- mice. In some of the latter tumors, one area of the tumor displayed loss of the Msh2+ allele, but not of the Apc+ allele, whereas another area displayed the inverse genotype. This apparent biclonality might indicate a requirement for collaboration between independent tumor clones during intestinal tumorigenesis.

Selective radiosensitization of drug-resistant MutS homologue-2 (MSH2) mismatch repair-deficient cells by halogenated thymidine (dThd) analogues: Msh2 mediates dThd analogue DNA levels and the differential cytotoxicity and cell cycle effects of the dThd analogues and 6-thioguanine.

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2+/+ and Msh2-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2+/+ and Msh2-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2+/+ and human HEC59/2-4 (MSH2+) MMR-proficient cells, whereas Msh2-/- cells and human HEC59 (MSH2-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2+/+ ES-derived and HEC59/2-4 cells to IdUrd, whereas Msh2-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2+ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2+/+ and Msh2-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G2-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.

Role of different genes in the virulence and pathogenesis of Aujeszky's disease virus.

In this study the role of different genes located in the unique short region of the genome of Aujeszky's disease virus was examined. Inactivation of the genes encoding the protein kinase (PK), gp63, and gI reduced virulence of the virus for pigs, in contrast to inactivation of the genes encoding the 28 kDa protein, and gX. There was no correlation between virulence and virus multiplication in vitro or in the oropharynx in vivo. The morphogenesis of the PK mutant was altered. The gI mutant replicated to normal titres in the oropharynx and could be recovered from the trigeminal ganglia but not from other parts of the central nervous system, suggesting that gI facilitates the spread of the virus from neuron to neuron. All mutants induced neutralizing antibody and complete or partial protection against a challenge infection. PK and gp63 were required for the induction of complete protection, although these proteins are reportedly not targets for neutralizing antibody or cytotoxic T cells.

Saturating mutagenesis and characterization of a herpesvirus genome using in vivo reconstitution of virus from cloned subgenomic regions.

The study of genome structure and gene function is pivotal in understanding the mechanisms of replication, pathogenesis, and virulence of herpesviruses. In this respect, mutagenesis and sequence analysis of genes encoded by the virus are of great importance. However, the herpesvirus genomes are large, with sizes ranging between 120 and over 200 kbp and encoding between 70 and 200 genes (see ref. 1 for a review). This large size hampers handling and systematic mutagenesis of the virus genome using standard modern molecular biology techniques. Most current methods of mutagenesis therefore do not rely on direct modification of the viral genome in vitro but depend on exchange in vivo, by homologous recombination, of a viral gene by a copy of the latter gene that is truncated in vitro by insertion of a marker gene. Mutant virus progeny can be screened or selected for, depending on the marker gene that is used. Commonly used marker genes are thymidine kinase and lacZ. This procedure is generally used, reliable, and has yielded a wealth of information on the function of herpers simplex virus type 1 (HSV-1) encoded genes. However, it requires prior mapping and cloning of every gene to be mutagenized and is therefore less feasible if the virus is a novel or less-well-known herpesvirus.

Macrophages as potential targets for zoledronic acid outside the skeleton-evidence from in vitro and in vivo models.

PURPOSE: Multiple cell types of the tumour microenvironment, including macrophages, contribute to the response to cancer therapy. The anti-resorptive agent zoledronic acid (ZOL) has anti-tumour effects in vitro and in vivo, but it is not known to what extent macrophages are affected by this agent. We have therefore investigated the effects of ZOL on macrophages using a combination of in vitro and in vivo models. METHODS: J774 macrophages were treated with ZOL in vitro, alone and in combination with doxorubicin (DOX), and the levels of apoptosis and necrosis determined. Uptake of zoledronic acid was assessed by detection of unprenylated Rap1a in J774 macrophages in vitro, in peritoneal macrophages and in macrophage populations isolated from subcutaneously implanted breast cancer xenografts following ZOL treatment in vivo. RESULTS: Exposure of J774 macrophages to 5 µM ZOL for 24 h caused a significant increase in the levels of uRap1A, and higher doses/longer exposure induced apoptotic cell death. DOX (10 nM/24 h) and ZOL (10 µM/4 h) given in sequence induced significantly increased levels of apoptotic cell death compared to single agents. Peritoneal macrophages and macrophage populations isolated from breast tumour xenografts had detectable levels of uRap1A 24 h following a single, clinically achievable dose of 100 µg/kg ZOL in vivo. CONCLUSION: We demonstrate that macrophages are sensitive to sequential administration of DOX and ZOL, and that both peritoneal and breast tumour associated macrophages rapidly take up ZOL in vivo. Our data support that macrophages may contribute to the anti-tumour effect of ZOL.

Multivariate analysis of MLH1 c.1664T>C (p.Leu555Pro) mismatch repair gene variant demonstrates its pathogenicity.

Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a "variant of uncertain significance" (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.

Multidrug resistance in breast cancer: from in vitro models to clinical studies.

The development of multidrug resistance (MDR) and subsequent relapse on therapy is a widespread problem in breast cancer, but our understanding of the underlying molecular mechanisms is incomplete. Numerous studies have aimed to establish the role of drug transporter pumps in MDR and to link their expression to response to chemotherapy. The ATP-binding cassette (ABC) transporters are central to breast cancer MDR, and increases in ABC expression levels have been shown to correlate with decreases in response to various chemotherapy drugs and a reduction in overall survival. But as there is a large degree of redundancy between different ABC transporters, this correlation has not been seen in all studies. This paper provides an introduction to the key molecules associated with breast cancer MDR and summarises evidence of their potential roles reported from model systems and clinical studies. We provide possible explanations for why despite several decades of research, the precise role of ABC transporters in breast cancer MDR remains elusive.

Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity.

We have mutagenized and mapped the gene encoding the large subunit of ribonucleotide reductase (RR1) in pseudorabies virus (PRV; synonyms Aujeszky's disease virus, suid herpesvirus type 1). PRV strains carrying an oligonucleotide that leads to termination of translation of the RR1 gene are avirulent for mice. We subsequently constructed a PRV strain carrying a deletion in the RR1 gene and also a PRV strain carrying both the deletion in the RR1 gene and a deletion in the glycoprotein g1 gene, which is a marker for PRV virulence. Both PRV strains were assayed for virulence and immunogenicity in pigs, the natural host for PRV. In contrast to a marker-rescued PRV strain, these RR1-deleted mutants were avirulent, were shed in very low titres in the oropharyngeal fluid by the animals, and induced low titres of neutralizing antibodies. However, protection against clinical signs after infection with virulent PRV was induced by both RR1-deleted mutants. The relative importance of viral RR and thymidine kinase enzymes for deoxynucleotide synthesis in viral replication is discussed. In addition, we discuss the potential use of RR as a target for anti-herpesviral drugs and the use of PRV strains, deleted for the RR1 gene, as vaccine strains.

Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells.

We have performed large-scale random oligonucleotide insertion mutagenesis on a 41-kbp genomic segment derived from the unique long (UL) region of the alphaherpesvirus pseudorabies virus (PRV). This procedure has resulted in the generation of a series of PRV strains, each carrying a single gene whose termination of translation is induced by the inserted oligonucleotide. To relate the genes that were involved in the mutagenization to genes previously identified in herpes simplex virus type 1, the prototype alphaherpesvirus, we have performed cross-hybridization studies. In this way, we have mapped the location of the homolog of a gene which was described to have sequence characteristics of a eukaryotic phosphotransferase. We characterized the phenotype of a mutant PRV strain lacking this putative phosphotransferase also the phenotype of a PRV strain lacking, in addition to the UL-encoded putative phosphotransferase, the protein kinase encoded within the unique short region of the virus. To assess the enzymatic activity of the UL region-encoded phosphotransferase, we expressed the gene transiently in a eukaryotic expression system. Immunoprecipitation of the protein followed by kinase assays and phosphoamino acid analyses revealed protein-serine/threonine kinase activity. Implications of sequence divergence of this protein from classical protein-serine/threonine kinases for kinase structure and function are discussed in view of the recent resolution of the structure of the catalytic domain of cyclic AMP-dependent protein kinase.

Site-directed mutagenesis of the Escherichia coli chromosome near oriC: identification and characterization of asnC, a regulatory element in E. coli asparagine metabolism.

We developed a new method for the specific mutagenization of the E. coli chromosome. This method takes advantage of the fact that a pBR322 plasmid containing chromosomal sequences is mobilizable during an Hfr-mediated conjugational transfer, due to an homologous recombination between the E. coli Hfr chromosome and the pBR322 derivative. Transconjugants are screened with a simple selection procedure for integration of mutant sequences in the chromosome and loss of pBR322 sequences. Using this method we specifically inactivated several genes near the E. coli replication origin oriC. We found that a gene coding for asparagine synthetase A. This regulatory mechanism was investigated in detail by determining in vivo regulation of asnA promoter activity by the 17kD protein under different growth conditions. Results obtained also suggest a general regulatory role of the 17kD protein in E. coli asparagine metabolism. Therefore the 17kD gene is proposed to be renamed asnC.

Identification of two genes in the unique short region of pseudorabies virus; comparison with herpes simplex virus and varicella-zoster virus.

We have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2.7 kb mRNA encoding the putative protein kinase and a 1.2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5' ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (greater than 95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mrs of 53K and 41K, and the 28K gene into a single protein with an Mr of 28K.

Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus.

We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M. van Zijl, W. Quint, J. Briaire, T. de Rover, A. Gielkens, and A. Berns, J. Virol. 62:2191-2195, 1988). This technique was used to construct, in a single experiment, a set of 13 viable pseudorabies virus strains with oligonucleotide insertions within all known genes of the Us region except for the gp50 gene, which proved essential for virus growth in cell culture. The growth rate in porcine kidney cells of mutants of all nonessential Us genes was similar to that of the parental virus, with the exception of a mutant of the recently identified protein kinase gene.

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.

Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion.

To investigate the function of the envelope glycoproteins gp50 and gII of pseudorabies virus in the entry of the virus into cells, we used linker insertion mutagenesis to construct mutant viruses that are unable to express these proteins. In contrast to gD mutants of herpes simplex virus, gp50 mutants, isolated from complementing cells, were able to form plaques on noncomplementing cells. However, progeny virus released from these cells was noninfectious, although the virus was able to adsorb to cells. Thus, the virus requires gp50 to penetrate cells but does not require it in order to spread by cell fusion. This finding indicates that fusion of the virus envelope with the cell membrane is not identical to fusion of the cell membranes of infected and uninfected cells. In contrast to the gp50 mutants, the gII mutant was unable to produce plaques on noncomplementing cells. Examination by electron microscopy of cells infected by the gII mutant revealed that enveloped virus particles accumulated between the inner and outer nuclear membranes. Few noninfectious virus particles were released from the cell, and infected cells did not fuse with uninfected cells. These observations indicate that gII is involved in several membrane fusion events, such as (i) fusion of the viral envelope with the cell membrane during penetration, (ii) fusion of enveloped virus particles with the outer nuclear membrane during the release of nucleocapsids into the cytoplasm, and (iii) fusion of the cell membranes of infected and uninfected cells.