Comparison of the therapeutic efficacy of 211At- and 131I-labelled monoclonal antibody MOv18 in nude mice with intraperitoneal growth of human ovarian cancer.

The purpose of the present study was to compare the therapeutic efficacy of the alpha-emitter Astatine-211 with the beta-emitter Iodine-131 bound to the specific monoclonal antibody MOv18. The measurements were performed in an ovarian cancer cell line (NIH:OVCAR 3) growing intraperitoneally in nude mice. Two weeks after the intraperitoneal inoculation of 1 x 10(7) cells of the human ovarian cancer cell line NIH:OVCAR-3 twenty mice were treated intraperitoneally with the specific monoclonal antibody MOv-18 labelled with either 211At (310-400 kBq) or 131I (5100-6200 kBq). The pharmacokinetics and biodistribution of labelled antibody in tumour-free animals were studied and the resulting bone marrow dose was estimated. When the mice were treated with 211At-labelled antibody 9 out of 10 mice were free of macro- and microscopic tumour compared to 3 out of 10 when Iodine-131 was used. The equivalent dose to the bone marrow was 2.4-3.1 Sv from 211At- and 3.4-4.1 Sv from 131I-irradiation. The therapeutic efficacy of 211At-labelled specific antibody is very good and, at approximately equivalent bone marrow doses, better than that of 131I.

The curative and palliative potential of the monoclonal antibody MOv18 labelled with 211At in nude mice with intraperitoneally growing ovarian cancer xenografts--a long-term study.

The purpose of the present study was to investigate the therapeutic efficacy of 211At-labelled specific monoclonal antibody MOv18 in nude mice with intraperitoneal growth of the human ovarian cancer cell line OVCAR3. In the first part of the study the antibody was injected intraperitoneally when the cancer growth was microscopic. The injected activity was 485-555 kBq. The median survival for treated mice was 213 days compared to 138 days for untreated mice (p < 0.014, log-rank test). No obvious toxicity was seen. Thirty-three percent of the mice were apparently free of cancer after 7 months and were probably cured. In the second part of the study mice with macroscopic cancer and signs of ascites were injected intraperitoneally with the same 211At-labelled antibody (377-389 kBq). This treatment possibly delayed the production of ascites. Hopefully radioimmunotherapy with regionally administered 211At-labelled antibody will be of value in women with ovarian cancer as well.

Radioimmunotherapy of nude mice with intraperitoneally growing ovarian cancer xenograft utilizing 211At-labelled monoclonal antibody MOv18.

The aim of this study was to investigate the therapeutic efficacy of 211At-labelled monoclonal antibody given intraperitoneally to nude mice with intraperitoneal growth of a human ovarian cancer cell line. Female nude mice were inoculated intraperitoneally with 1 x 10(7) cells of the human ovarian cancer cell line NIH:OVCAR 3. After about two weeks they were injected with the 211At-labelled specific monoclonal antibody MOv18 intraperitoneally. For comparison, other groups of mice were given the same labelled antibody intravenously, 211At-labelled unspecific antibody C242 intraperitoneally or unalbelled MOv18 intraperitoneally. Six weeks later the animals were sacrificed and the occurrence of tumour and ascites was determined. When the mice were treated with 211At-labelled MOv18 intraperitoneally 9 out of 10 were apparently free of both ascites and tumour compared to none of the mice given unlabelled antibody. 211At-labelled MOv18 given intravenously or 211At-labelled unspecific antibody given intraperitoneally were less effective. Regional radioimmunotherapy with the alpa-emitter 211Astatine seems to be an effective treatment of nude mice with intraperitoneally growing human ovarian cancer. Hopefully this treatment can be given in an adjuvant setting to women with minimal residual ovarian cancer in the future.

Exon deletions and variants of human estrogen receptor mRNA in endometrial hyperplasia and adenocarcinoma.

Estrogen receptors (estrogen receptor alpha, ER) belong to a family of ligand-modulated transcription factors that play an important role in the progression of such tumors as breast and endometrial cancers. Functional domains, a set of mutations and variants produced by internal deletions of ER mRNA, have mainly been identified in breast cancer. Experimental results suggest that the presence of variants may result in different proteins which differ in activity and modulate the ER signaling pathway differently. We analyzed samples from 21 cases of endometrial hyperplasia and from 29 cases of endometrial cancer for the presence of internal exons and exon deletion variants of ER mRNA. ER and progesterone receptor (PgR) proteins were measured using Western blot technique in all endometrial cancer samples. We found that absence of the wild-type exon PCR product of ER mRNA in a sample increased in parallel with malignant potential in both sample types, whereas the number of exon deletion variants detected in the same sample decreased in cases of malignancy. The precise deletions of the respective exons suggest that they are probably the result of splicing errors. A relatively high number of variants in hyperplasia samples may indicate the important role of ER mRNA variants in the physiologic regulation of transcription in estrogen-sensitive genes. Eleven of 29 adenocarcinomas expressed a 62-kDa ER protein, truncated at the amino terminal, whereas all but one sample expressed a short 52 kDa variant ER protein. Our results suggest that differing ER proteins are generally present in human endometrial adenocarcinomas and that they may influence the estradiol signaling pathways.

The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging.

Deletion of the cytoplasmic tails of the influenza A virus spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), has previously been shown to result in markedly defective virion morphogenesis (Jin et al., 1997, EMBO J. 16, 1236-1247). We have found that influenza A virus preparations lacking the HA and NA cytoplasmic tails (HAt-/NAt-) have a reduced vRNA to protein content, contain an increase in cellular RNA contaminants, and exhibit increased resistance to ultraviolet (UV) inactivation. There is also a direct correlation between abnormal virion morphology and reduced infectivity. The data suggest that the HAt-/NAt- virion population contains a broader range of number of packaged RNA segments than wild-type (wt) virus. Sucrose gradient centrifugation analysis indicated the presence of a subpopulation of virions with pronounced deformation in virion morphology and reduced infectivity. The role of the HA and NA cytoplasmic tails was examined further by using a trans-complementation assay and it was found that expression of wt HA and NA from cDNAs followed by HAt-/NAt- virus infection caused the formation of a pseudotype virus with wt sedimentation properties. Taken together the data indicate that the HA and NA cytoplasmic tails affect not only virion morphology but also proper genome packaging.

The signal for clathrin-mediated endocytosis of the paramyxovirus SV5 HN protein resides at the transmembrane domain-ectodomain boundary region.

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is internalized from the cell surface via clathrin-coated pits. However, the cytoplasmic domain of SV5 HN does not contain a previously characterized internalization motif. A cell-surface-expressed chimeric protein (APK), consisting of the cytoplasmic tail, transmembrane (TM) domain, and 12 residues of the ectodomain of HN joined to the cytoplasmic protein pyruvate kinase is internalized, indicating that the N-terminal region of HN contains an internalization signal. Although SV5 HN is internalized at a rate similar to that of influenza virus hemagglutinin (HA) mutant Y543, which contains a degenerate tyrosine-based signal in its cytoplasmic tail, the elimination of the majority of the HN cytoplasmic tail, or substitution of the HN TM domain with leucine residues, did not affect the rate of HN internalization. The HN protein of the closely related virus, Newcastle disease virus (NDV), is not internalized from the cell surface. Working under the usual convention that the TM domain consists of the hydrophobic residues bounded by two charged residues, analysis of internalization of mutant and chimeric NDV HN molecules indicates that the first seven SV5 HN ectodomain residues are critical for internalization of HN. A glutamic acid residue (E37) that abuts this presumptive HN TM domain/ectodomain boundary is important for SV5 HN internalization.

Biokinetics of the monoclonal antibodies MOv 18, OV 185 and OV 197 labelled with 125I according to the m-MeATE method or the Iodogen method in nude mice with ovarian cancer xenografts.

The biodistribution of the radiolabelled monoclonal antibodies MOv18, OV185 and OV197 in nude mice with subcutaneous tumours of the human ovarian cancer cell line OVCAR3 was investigated. The early uptake of MOv18 (1-24 h) and the uptake in relation to tumour size were also studied. The antibodies were labelled with 125I according to the Iodogen method or the m-MeATE method, the latter also being suitable for labelling with 211Astatine. The tumour/blood ratio and the localization index for Mov 18 72 h after antibody injection were 2.21 +/- 0.25 and 4.62 +/- 1.27, respectively. This is significantly higher than for the other two specific antibodies. The early tumour uptake of MOv18 was low with a tumour/blood ratio of 0.23 +/- 0.04 after 6 h, and the uptake was higher in small tumours. The two labelling methods were found to be equivalent. We conclude that MOv18 labelled according the m-MeATE method should be suitable for further therapeutic studies with 211Astatine in nude mice.

Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation.

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, alpha-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998). Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an alpha helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.

The paramyxovirus SV5 small hydrophobic (SH) protein is not essential for virus growth in tissue culture cells.

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.

Estradiol induces DNA fragmentation in a human endometrial adenocarcinoma with estradiol-inhibited growth phenotype.

A moderately differentiated human endometrial adenocarcinoma heterotransplanted into nude mice was investigated for morphological and molecular changes in the tumours after treating the animals with estradiol. The tumour growth was previously characterised as estradiol-independent but responsive (inhibited) without any changes in cell proliferation. In response to hormonal treatment rather the cell loss factor increased. In this experiment tumours influenced by estradiol were investigated at different time-points after treatment by an in situ labelling technique to detect cells undergoing DNA fragmentation as a sign of apoptosis. Expression of the apoptosis related protein bcl-2 was evaluated by Western blotting. Tumours from animals treated with estradiol showed an increase in tumour volume doubling time from 5.4 days to 16 days compared to control tumours. Histologically, tumours influenced by estradiol were better differentiated than control tumours and showed a significant increase in cells staining positively with the in situ apoptosis detection technique. A parallel time dependent decreased expression of bcl-2 protein was observed. These results confirm our previous findings where estradiol influenced the cell loss factor without changes in the growth fraction, indicating increased apoptotic activity in response to hormonal treatment.

Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape.

The cytoplasmic tails of the influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA) are highly conserved in sequence for all virus subtypes and it is believed that assembly of this enveloped virus depends on interactions of these domains with cytoplasmic viral components. However, it is possible to rescue altered influenza viruses lacking either the HA or NA cytoplasmic tails. We have obtained an influenza virus that lacks both the cytoplasmic tail of HA and NA. Particle production is reduced approximately 10-fold but these particles, although having a fairly normal protein composition, are greatly elongated and of extended irregular shape. We propose a model in which the interactions of the cytoplasmic tails of HA and NA with an internal viral component are so important for spherical virion shape that there is dual redundancy in the interactions.

Tumour growth and cell kinetics in variants of a human endometrial adenocarcinoma expressing either wild-type or mutant p53.

We have compared the baseline cell proliferation and tumour growth in two variants of a human endometrial adenocarcinoma grown in nude mice. One of these tumour variants expressed wild-type p53 whereas the other had mutations of the p53 gene at codon 175 in both alleles and at codon 248 in one allele. There was no difference in growth rate between the tumour variants. Cell proliferation parameters, such as labelling index and S-phase fraction, were significantly increased in the tumour with mutated p53 and consequently there was a significantly lower proportion of cells in the G1-phase, proposing an at least partial loss of suppressor function in this tumour. Semi-quantitative analysis of the p53 and bcl-2 proteins showed a significant overexpression of p53 and a decreased expression of the bcl-2 protein in the p53 mutated tumour variant compared with the variant with wild-type p53. We conclude that wild-type p53 protein acts as an active suppressor in the regulation of the baseline growth and cell kinetics of this tumour and could be linked through a p53--bcl-2 system in human endometrial adenocarcinomas.

Estradiol regulates tumor growth by influencing p53 and bcl-2 expression in human endometrial adenocarcinomas grown in nude mice.

This study analyzes the effects of estradiol on p53 and bcl-2 expression, tumor growth and cell kinetic parameters in three human endometrial adenocarcinomas grown in nude mice. The tumors used were estradiol receptor (ER) positive but differed in receptor concentration and hormone sensitivity. All three tumors expressed wild-type p53 protein. Using a tumor with an estradiol independent but responsive (inhibited) growth phenotype, we found that an increase in the circulating estradiol concentration led to increases in p53 expression and a decrease in bcl-2 levels, resulting in increased cell loss (CL) measured as delayed tumor growth. In another tumor which demonstrated estradiol independent and resistant growth, we observed an estradiol dose-related increase in p53 expression but no changes in bcl-2 expression or cell kinetic parameters. The ER mechanism of these cells was at least partly intact, as evidenced by maintained PgR induction. The third tumor showed an estradiol independent and resistant growth phenotype and a non-functional ER mechanism, lacking PgR induction. After estradiol treatment of the tumor-bearing animals no changes were observed in p53 or bcl-2 expression or in cell kinetics. We conclude that estradiol may regulate tumor growth in some ER positive human endometrial adenocarcinomas through regulation of p53 expression, which in turn regulates the bcl-2 protein concentration. Furthermore, this regulation of p53 expression is estradiol dose dependent. These growth regulating functions appear to be strongly influenced by ER mechanisms and do not seem to operate synchronously in tumors with an estradiol resistant growth phenotype.

Characterisation of a human endometrial adenocarcinoma established in vivo and in vitro.

A moderately differentiated human endometrial adenocarcinoma was heterotransplanted into nude mice and later established as a continuous in vitro cell line. Western blot analysis showed an accumulation of p53 protein in the cell line compared to the original tumour and heterotransplants. Sequential analysis of the p53 gene revealed point mutations in codons 175 and 248 in the cell line while no mutations were found prior to in vitro establishment. Immunohistochemistry confirmed the epithelial origin of the heterotransplants and of retransplants of the cell line. The intraperitoneal retransplants remained moderately differentiated, whereas subcutaneous retransplants became less differentiated. Heterotransplants were estrogen receptor (ER) positive and progesterone receptor (PgR) negative, indicating preservation of normal steroid receptor status. The ER could not be detected in the in vitro cell line using an enzyme immunoassay, but was detected with Western blot using a polyclonal antibody toward the carboxy terminus. After estradiol treatment, the in vitro cell line became weakly positive for the PgR, suggesting the ER mechanism was at least partly intact. Tumour growth in vivo was independent of endogenous estrogen but was inhibited when the tumour-bearing animals were treated with estradiol. Analysis of cell growth kinetics by flow cytometry (FCM) after bromodeoxyuridine (BrdU)-labelling revealed no difference in S-phase fraction (SPF) or labelling index (LI) between the treated and control groups. Cell loss (CL) was significantly increased from 42% to 89%, resulting in increased tumour volume doubling time (TVDT). Under in vitro conditions estradiol treatment resulted in an increase in cell doubling time and this growth retardation was accompanied by a significant decrease in SPF and LI. The estrogen responsive (inhibited) phenotype was thus preserved in the in vitro cell line but was probably mediated through another mechanism. This cell line thus appears to represent the development of a more malignant clone with divergent receptor function and growth behaviour, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.

Influenza B virus NB glycoprotein is a component of the virion.

The influenza B virus NB glycoprotein is abundantly expressed at the surface of virus-infected cells. NB spans the membrane once and has an 18 amino acid ectodomain, a 22 amino acid transmembrane domain, and a 60 amino acid cytoplasmic tail. The NB N-terminal ectodomain contains two asparagine residues that are modified by the addition of palmitic N-linked carbohydrate chains, which become further modified by the addition of polylactosaminoglycan. We have now shown that NB is also modified by addition of acid. To determine if NB is incorporated into virions, metabolic labeling, immunoblotting, and immunogold electron microscopy techniques were used. NB was identified in virions grown in MDCK cells or in embryonated chicken eggs in two forms: (a) NB modified by addition of polylactosaminoglycan (NBpl), and (b) a cleaved species (NBc) that has a smaller molecular weight than unglycosylated NB (NB12). Proteinase K digestion of purified virions converted NBpl to NBc. Examination of virions purified by isopycnic centrifugation by electronmicroscopy and immunogold staining, using an affinity-purified antibody raised to a peptide derived from the NB cytoplasmic tail, showed staining for NB in influenza B virions. Quantification of the amount of NB in purified virions using two unrelated biochemical methods indicated there are on average approximately 15-100 molecules of NB per virion. Although the number of NB molecules incorporated on average into an influenza B virus particle is small, this finding is reminiscent of the number of molecules (14-68 monomers) found on average of the M2 integral membrane protein of influenza A virus.

The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus.

High level expression of the M2 ion channel protein of influenza virus inhibits the rate of intracellular transport of the influenza virus hemagglutinin (HA) and that of other integral membrane glycoproteins. HA coexpressed with M2 is properly folded, is not associated with GRP78-BiP, and trimerizes with the same kinetics as when HA is expressed alone. Analysis of the rate of transport of HA from the ER to the cis and medial golgi compartments and the TGN indicated that transport through the Golgi apparatus is delayed. Uncleaved HA0 was not expressed at the cell surface, and accumulation HA at the plasma membrane was reduced to 75-80% of control cells. The delay in intracellular transport of HA on coexpression of M2 was not observed in the presence of the M2-specific ion channel blocker, amantadine, indicating that the Golgi transport delay is due to the M2 protein ion channel activity equilibrating pH between the Golgi lumen and the cytoplasm, and not due to saturation of the intracellular transport machinery. The Na+/H+ ionophore, monensin, which also equilibrates pH between the Golgi lumen and the cytoplasm, caused a similar inhibition of intracellular transport as M2 protein expression did for HA and other integral membrane glycoproteins. EM data showed a dilation of Golgi cisternae in cells expressing the M2 ion channel protein. Taken together, the data suggest a similarity of effects of M2 ion channel activity and monensin on intracellular transport through the Golgi apparatus.

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity.

The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.

Estradiol influences p53 expression in a human endometrial adenocarcinoma heterotransplanted into nude mice.

The influence of different estradiol concentrations on the expression of the p53 suppressor gene and on cell kinetics was examined by semiquantitative analysis of protein and bromodeoxyuridine labelling in a human endometrial adenocarcinoma grown in nude mice. We found that increasing the circulating estradiol increases (p = 0.001), and decreasing the hormone value decreases (p = 0.001) the expression of p53 in this tumor. The number of cells in the G1/G0 phase of the cell cycle was significantly higher (p = 0.03), and the number of cells in the G2/M phase was significantly lower (p = 0.01) in tumors grown in estradiol-treated mice than in tumors obtained from the nontreated group. Changes in p53 expression may possibly be explained by either altered transcription activity of the gene or increased half-life of the protein. Our results suggest an important role of estradiol in the progression of estrogen receptor (ER) positive human endometrial adenocarcinomas.

The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.

The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions.

The paramyxovirus simian virus 5 (SV5) cysteine-rich V protein has been shown to be a virus structural protein by analysis of the polypeptides of purified SV5 virions. In addition, the V protein has been identified as a component of the virus nucleocapsid core both by the analysis of the polypeptides present in radioactively labeled preparations of purified nucleocapsids and by immunoelectron microscopy. Quantitative autoradiography was used to determine that there are approximately 350 molecules of the V protein in virions. The V protein has been purified from V recombinant baculovirus-infected insect cells and by using inductively coupled argon plasma atomic emission spectroscopy it was found that each molecule of V binds two zinc atoms.