The polypeptide composition of vaccinia-infected cell membranes and rifampicin bodies.

The protein and glycoprotein composition of a sucrose gradient fraction from vaccinia infected cells treated with rifampicin was studied. This particulate fraction contained cytoplasmic membranes and pleomorphic membranous structures. The glycoproteins (89, 42 and 20-23 kDa, respectively) were identified as the same glycoproteins that are found in plasma membranes of infected cells and the envelope of extracellular enveloped vaccinia (EEV). These glycoproteins could be solubilized by 0.1% NP-40. The Golgi membrane associated 41K acylated vaccinia protein was also NP-40 soluble. In contrast, most particulate fraction proteins (125, 100, 86, 65, 41, 39, 31, 27, 25, 14 and 12.5 kDa) with the exception of the 33 and 29 kDa proteins remained essentially insoluble after NP-40 treatment. The 86 and 65 kDa proteins are the rifampicin inhibited precursors to INV core proteins while the 33 and 29 kDa proteins are INV surface proteins. Twelve proteins behaved like their respective comigrating INV proteins when extracted with NP-40 and 2ME. Electron microscopy showed that a centrifuged sediment from NP-40 treated cells contained pleomorphic protein containing membranous structures that we have called rifampicin bodies. We conclude that (1) the major glycoproteins found in the particulate fraction from sucrose gradients are vaccinia glycoproteins residing in cytoplasmic membranes while (2) the major non-glycosylated proteins are components of the rifampicin bodies and that (3) the rifampicin bodies represent an intermediate in the morphogenetic process leading to mature INV.

Epidermal powder immunization with a recombinant HIV gp120 targets Langerhans cells and induces enhanced immune responses.

The recombinant envelope gp120 (rgp120) of human immunodeficiency virus (HIV) is a weak immunogen when administered by intramuscular (IM) injection. In the present study, we report that epidermal powder immunization (EPI) elicits robust antibody responses to the rgp120. EPI of mice with a dose 0.2-5 microg of rgp120 protein elicited geometric mean antibody titers that were 18- to 240-fold higher than that elicited by IM injection using a 5.0 microg dose. Targeting antigen to and mobilization of Langerhans cells (LCs) by EPI may explain the enhanced immunogenicity of the rgp120. EPI with rgp120 using sugar and gold particles as carrier resulted in differential antigen entry into the LCs and differential IgG subclass antibody and cellular immune responses. EPI may serve as a useful tool to evaluate vaccine potential of the rgp120 protein.

Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions.

A new method of preparing ionically cross-linked polyphosphazene hydrogel microspheres which enables effective control over microsphere size distribution has been developed. The synthesized microspheres can be used in protein delivery and, especially, as vaccine delivery vehicles. A new technique utilizes the ability of aqueous solutions of poly[di(carboxylatophenoxy)phosphazene] (PCPP) to form coacervate microdroplets upon addition of sodium chloride. These microdroplets are then stabilized via polymer cross-linking with calcium ions. It was demonstrated that the method enables efficient microencapsulation of proteins. It was also shown that microsphere size can be controlled through the manipulation of microencapsulation conditions. The process is simple, highly reproducible and generates microspheres with a narrower microsphere size distribution, compared to the previous technologies.

Synthesis of N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline (lisinopril).

The synthesis and some of the spectral properties of N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline (lisinopril, MK-521) are described. This compound inhibits angiotensin-converting enzyme with an IC50 of 1.2 X 10(-9) M.

Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia.

The significance of extracellular enveloped vaccinia (EEV) for the in vitro and in vivo dissemination of vaccinia virus was investigated. The quantity of in vitro released extracellular virus correlated very closely with the ability of 13 vaccinia strains to cause long-range spread of infection (comet formation) in cell cultures infected at low m.o.i. but was not correlated with plaque size. The kinetics of virus spread after low m.o.i. was related to the amount of virus released from primary infected cells but not to their content of intracellular naked vaccinia (INV). Most extracellular vaccinia virus from IHD-J-infected RK-13 cells banded in CsCl density gradients as EEV (88%) while very little banded as INV (2%). Antisera to the enveloped prevented comet formation while antisera to INV did not. CsCl centrifugation of blood-borne extracellular virus from rabbits infected intravenously with vaccinia virus after cyclophosphamide treatment revealed that 64% of the virus banded as EEV but only 11% as INV. High in vitro EEV-yielding vaccinia strains were able to spread from the respiratory tract to the brains of mice and cause death. Low in vitro EEV-yielding vaccinia strains were generally not able to disseminate in vivo or cause mouse mortality. The notable exception to this trend was strain WR, which, although releasing small amounts of virus in vitro, could nevertheless very effectively disseminate in vivo, causing a high rate of mouse mortality. Treatment with anti-envelope serum protected mice from a lethal vaccinia infection whereas antiserum to inactivated INV did not. These results indicate that the in vitro dissemination of vaccinia infection is mediated by EEV and implicate EEV as having a role in the in vivo dissemination.

Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus.

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.

The existence of an envelope on extracellular cowpox virus and its antigenic relationship to the vaccinia envelope.

Virus released from cowpox infected cells was demonstrated by electron-microscopy to be surrounded by an envelope not present on mature intracellular virus. Enveloped cowpox had an isopycnic density of 1.23 g/ml, was infectious and neutralized by antibodies specific for the envelope antigens of vaccinia virus but not neutralized by antibodies specific for intracellular naked vaccinia virus. Only 1 per cent of the total intracellular virus yield was released as enveloped virus. The major cowpox glycoprotein (76 K) and a 44 K glycoprotein did not comigrate with any vaccinia glycoproteins whereas cowpox glycoproteins at 42 K and 20-23 K did coelectrophores with vaccinia glycoproteins. All of the mentioned cowpox glycoproteins were precipitated by vaccinia antiserum.

Characterization of vaccinia virus glycoproteins by monoclonal antibody precipitation.

Monoclonal antibodies were used to characterize vaccinia virus glycoproteins known to be incorporated into the envelope of extracellular enveloped vaccinia (EEV) virus. The 89K hemagglutinin, 42K, and 23-28K glycoproteins were predominantly expressed as late vaccinia proteins. The 89K glycoprotein was sulfated and phosphorylated but not acylated. 89K precursor proteins of 32K, 41.5K, and 52K were detected. The former had a molecular weight expected from the deduced amino acid sequence of the hemagglutinin gene. A 76K glycoprotein that did not contain methionine and was not sulfated or phosphorylated was precipitated late in infection by the anti-hemagglutinin monoclonal antibody. The appearance of this protein was inhibited by rifampicin and it may thus result from 89K cleavage. A 220K complex contained some or all of the hemagglutinin gene products linked by disulfide bonds. The 42K glycoprotein was not sulfated or phosphorylated but was acylated. This glycoprotein was disulfide bonded with the EEV 37K nonglycosylated envelope protein. The 23-28K glycoprotein was not sulfated but was both phosphorylated and acylated. The 23-28K glycoprotein group of five proteins had a common protein backbone that was differentially glycosylated. Pulse-chase, glycosylation inhibition with tunicamycin, and glycosidase experiments established that the precursor to the 23-28K glycoproteins was a 21K protein. Members of this protein family formed dimers of approximately 55K through disulfide bonds.

Adsorption and penetration of enveloped and naked vaccinia virus particles.

The adsorption and penetration of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) were examined. The adsorption kinetics of INV and EEV were similar, but INV adsorption was found to be more sensitive to the adsorption environment than EEV. The PFU-to-particle ratio for the two virus particles indicated that EEV was approximately two times as infectious as INV. Kinetic studies at 37 degree C showed that EEV penetrated cells more rapidly than INV. Penetration of EEV was unaffected by incubation in phsophate-buffered saline, but was somewhat reduced by incubation at 22 degree C. In contrast, INV penetration was effectively eliminated by incubation in phosphate-buffered saline or by incubation at 22 degree C. In addition, INV but not EEV pentration was sensitive to treatment with sodium fluoride and cytochalasin. B. These results are discussed with regard to the mechanism of INV and EEV penetration.

Mechanism of vaccinia virus release and its specific inhibition by N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine.

The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.

Extracellular release of enveloped vaccinia virus from mouse nasal epithelial cells in vivo.

The release of vaccinia virus from mouse nasal epithelial cells infected in vivo was studied by electron microscopy. Intracellular naked vaccinia virus was enwrapped by Golgi membranes to form a double membrane intermediate. The outer membrane of the intermediate presumably fused with the plasma membrane, releasing extracellular enveloped virus. No signs of simple naked virus budding at the plasma membrane were observed. The majority of extracellular virus was enveloped and not naked.

The effect of cytochalasin D and monensin on enveloped vaccinia virus release.

Both monensin and cytochalasin D reduced the production of infectious cell associated virus and infectious extracellular virus with the latter clearly being the more sensitive. The difference in yields were even more clearly seen if the yield of virus particles was monitored instead of yield of infectious virus. Addition of 1 microgram/ml cytochalasin D or 1 microM monensin to the growth medium of vaccinia virus-infected cells inhibited the appearance of extracellular enveloped vaccinia virus (EEV) in the growth medium without affecting the production of intracellular naked vaccinia virus (INV) particles. Although EEV was not released into the medium of cytochalasin D treated cells, EEV was, nevertheless, detectable in CsCl gradients of cell associated virus. Monensin treatment did not affect the synthesis of vaccinia glycoproteins but did significantly reduce the transport of these glycoproteins to the cell surface and also reduced the secretion of proteins. Monensin had the additional effect of causing the appearance of numerous large vacuoles in the cytoplasm. Vaccinia is normally wrapped by cytoplasmic membranes in preparation for release. The monensin induced conversion of cytoplasmic membranes to large vacuoles is presumably the basis for the block in virus wrapping and subsequent release. Cytochalasin D did not alter any of the steps in protein synthesis, transport or secretion. Electronmicroscopic studies confirmed the existence of EEV on the surface of infected cells treated with cytochalasin D. This drug which specifically affects cellular microfilament organisation thus imposes a block on the final release of EEV from the cell surface.

Effect of glycosylation inhibitors on the release of enveloped vaccinia virus.

Addition of 1 to 10 mM 2-deoxy-D-glucose (2-dg) or glucosamine (gln) to the growth medium of vaccinia virus-infected cells inhibited the release of extracellular enveloped vaccinia virus (EEV) without affecting the production of intracellular naked vaccinia virus (INV) particles. In contrast, INV infectivity (particles per PFU) was decreased sevenfold by 50 mM 2-dg. Treatment with 2-dg reduced but did not eliminate glycosylation of the INV 37,000-molecular-weight glycoprotein. The kinetics of sensitivity to inhibitor addition experiments and inhibitor reversal experiments indicated that EEV release was dependent on glycosylation before 8 h postinfection. This was supported by polyacrylamide gel electrophoretic analysis of the synthesis kinetics for cell membrane-associated vaccinia glycoproteins in 2-dg-inhibited infected cells. The dependence of vaccinia protein glycosylation before 8 h postinfection for efficient EEV release was observed in spite of the fact that the period of greatest glycoprotein synthesis was 8 to 12 h postinfection. The presence of 2-dg resulted in an incompletely glycosylated 89,000-molecular-weight glycoprotein, as indicated by a reduction in the apparent glycoprotein molecular weight. The morphological event affected by the inhibitors was the acquisition by INV of a double-membrane structure from the Golgi apparatus. This morphological intermediate is necessary for release of EEV.

Presence of haemagglutinin in the envelope of extracellular vaccinia virus particles.

The relationship of vaccinia haemagglutinin (HA) to extracellular enveloped virus (EEV) was examined. EEV banded in caesium chloride gradients at a density of 1.23 to 1.24 g/ml coincident with a peak of HA activity. EEV of an HA+ vaccinia strain showed greater than 90% adsorption to rooster red blood cells (RBCs) as detected by infectivity and 3H-thymidine labelling whereas intracellular naked virus (INV) of the HA+ strain and EEV of an HA- strain failed to show significant adsorption. The adsorption was specifically inhibited by antiserum to vaccinia. Adsorption kinetic experiments demonstrated a lack of temperature dependence on the total amount of EEV adsorbed. No elution of EEV from RBCs could be detected. The capacity of EEV to adsorb to RBCs was found to be stable at 56 degrees C for 30 min.

Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein "footprinting".

This study describes the use of limited proteolysis of monoclonal antibody (mAb)-bound antigens in the analysis of the two measles virus surface glycoproteins. This approach is dubbed protein "footprinting" in analogy with DNA "footprinting." Protein footprinting was superior to competitive-binding assays and as good as in vitro mAb-selected variant analysis in differentiating among mAbs with various specificities to a given protein. Proteolytic digestion of the antigen prior to mAb binding drastically reduced mAb binding resulting in poor differentiation among mAbs. In contrast, protein footprinting showed that some mAbs retained the ability to immunoprecipitate such fragments. Thus footprinting could be used for localization of mAb epitopes on a protein and proved also to be an effective means of distinguishing among mAb-selected variants differing in single epitopes. Conformational changes caused by heat-denaturation or the binding of anti-antibody to an antigen-antibody complex could also be detected by footprinting.

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.