Alteration of amino acid 101 within capsid protein VP-1 changes the pathogenicity of Theiler's murine encephalomyelitis virus.

Chronic Theiler's murine encephalomyelitis virus infection of susceptible mice is an animal model for human demyelinating diseases. Previously we described an altered and diminished pattern of central nervous system disease in immunocompetent SJL/J mice infected with a variant virus. This variant virus H7A6-2 was selected with a neutralizing mAb recognizing the capsid protein VP-1 of Theiler's virus. Here we characterize the variant virus by ELISA and neutralization assays and by sequencing selected regions of the viral RNA genome and relate the alteration to disease. The variant virus contains one single point mutation within a neutralizing epitope of VP-1. This nucleotide change lead to an amino acid replacement at amino acid 101 of VP-1, a threonine (wild type) to an isoleucine (variant). Model building based on sequence alignments and the known structure of the related Mengo virus indicates that the altered amino acid is located in an exposed loop on the surface of the virus at the periphery of a site that has been proposed to be the receptor binding site. The results of ELISA, neutralization assay, and direct RNA sequencing provide for the first time an opportunity to precisely map an important structural determinant of neurovirulence.

The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host.

An immunohistochemical study was conducted to detect the temporal infection sequence of Autographa californica M nuclear polyhedrosis virus in Trichoplusia ni larvae. Staining patterns indicated that the initial infection occurred in the midgut, simultaneously in columnar epithelial and regenerative cells, but that subsequently this tissue recovered. A major envelope glycoprotein stained in a polar fashion when it was expressed in columnar epithelial cells, but not when expressed in other cells types. Systemic infection was mediated by free virus for some tissues whereas infected hemocytes appeared to spread virus to other tissues by an unknown mechanism. A cell to cell spread within several tissues was detected. These results have important implications for baculoviruses engineered for improving their pesticide potential.

Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines.

Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex.

We studied the maturation of Uukuniemi virus and the localization of the viral surface glycoproteins and nucleocapsid protein in infected cells by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy with specific antisera prepared in rabbits against the two glycoproteins G1 and G2 and the nucleocapsid protein N. Electron microscopy of thin sections from infected cells showed virus particles maturing at smooth-surfaced membranes close to the nucleus. Localization of the G1/G2 and N proteins by indirect immunofluorescence at different stages after infection showed the antigens to be present throughout the cell interior but concentrated in the juxtanuclear region. The G1/G2 antiserum also appeared to stain the nuclear and plasma membranes. Double staining with tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, which preferentially stains the Golgi complex, and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, which stained the G1/G2 or N proteins, showed that the staining of the juxtanuclear region coincided. Similarly, double staining for thiamine pyrophosphatase, an enzyme activity specific for the Golgi complex, showed the fluorescence and the cytochemical stain to coincide in the juxtanuclear region. Immunoperoxidase electron microscopy of cells permeabilized with saponin revealed that the viral glycoproteins were present in the rough endoplasmic reticulum and the nuclear and Golgi membranes; the latter was heavily stained. With this method, the N protein was localized to the cytoplasm, especially around smooth-surfaced vesicles in the Golgi region. Taken together, the results indicate that Uukuniemi virus and its structural proteins accumulate in the Golgi complex, supporting the idea that this compartment rather than the plasma membrane is the site of virus maturation. This raises the interesting possibility that deficient transport of the glycoproteins to the plasma membrane and hence their accumulation in the Golgi complex determines the site of virus maturation.

Natural and experimentally induced antibodies to defined mammalian type-C virus proteins in primates.

Using sensitive radiommunoprecipitation assays for highly purified type-C RNA tumor virus proteins, we found that 5 of 16 clinically normal gibbons (including 4 of 5 normal animals from a colony with 2 cases of lymphoma) and 4 of 4 experimentally inoculated gibbons formed antibodies to the major structural protein (p30) of gibbon ape leukemia virus (GaLV). An additional woolly monkey immunized with the closely related simian sarcoma virus also formed antibodies detectable with GaLV p30. Of 20 patients immunized with formalin-inactivated Rauscher murine leukemia virus (R-MuLV), 10 were previously reported to have antibodies to MuLV as determined by an internally labeled banded virus radioimmunoprecipitation assay. In comparison studies with purified R-MuLV proteins, 7 of 20 patients formed antibodies: 3/20 to R-MuLV p30 only, 1/20 to R-MuLV glycoprotein (gp) 70 only, and 3/20 to both p30 and gp70. Most responders were melanoma patients receiving immunotherapy with BCG. Additionally, rhesus monkeys produced antibodies to the endogenous cat virus RD114 and closely related endogenous baboon leukemia virus p30's. Thus these studies demonstrated the ability of primates (including humans) to form antibodies to well-characterized proteins from endogenous and exogenous type-C viruses and the potential utility of these assays for seroepidemiologic studies.

Growth of murine sarcoma virus-transformed rat kidney cells in nude mice: absence of induction of host endogenous viruses.

Clonal isolates of the normal rat kidney cell line (NRK) transformed by a defective murine sarcoma virus (Kirsten strain) were injected into nude mice of BALB/c background to determine whether the growth of these cells as tumors was accompanied by the induction of host endogenous type C viruses. All the virus-transformed clones produced rapidly growing tumors in nude mice, but neither the induction of mouse endogenous viruses nor the rescue and spread of the transforming sarcoma virus were observed during the growth of tumors. The degree of expression of the tumor virus structural proteins in the transformed cells did not determine the cellular phenotype with regard to tumorigenicity in nude mice, nor did it modify the cellular growth properties in vitro. Consistent with earlier observations with simian virus 40-transformed mouse and rat cells, the ability of sarcoma virus-transformed NRK cells to initiate tumor growth in nude mice appeared to be correlated with anchorage-independent growth in vitro.

Cofactors with human papillomavirus in a population-based study of vulvar cancer.

BACKGROUND: Human papillomavirus (HPV) has been previously associated with vulvar cancer. In a population-based study, we examined whether exposure to HPV, cigarette smoking, or herpes simplex virus 2 (HSV2) increases the risk of this cancer. METHODS: Incident cases of in situ (n = 400) and invasive (n = 110) squamous cell vulvar cancer diagnosed among women living in the Seattle area from 1980 through 1994 were identified. Serum samples were analyzed for antibodies against specific HPV types and HSV2. HPV DNA in tumor tissue was detected by means of the polymerase chain reaction. In most analyses, case subjects were compared with population-based control subjects (n = 1403). Relative risks of developing vulvar cancer were estimated by use of adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Increased risks of in situ or invasive vulvar cancer were associated with HPV16 seropositivity (ORs = 3.6 [95% CI = 2.6-4.8] and 2.8 [95% CI = 1.7-4.7], respectively), current cigarette smoking (ORs = 6.4 [95% CI = 4.4-9.3] and 3.0 [95% CI = 1.7-5.3], respectively), and HSV2 seropositivity (ORs = 1.9 [95% CI = 1.4-2.6] and 1.5 [95% CI = 0.9-2.6], respectively). When the analysis was restricted to HPV16 DNA-positive tumors (in situ or invasive), the OR associated with HPV16 seropositivity was 4.5 (95% CI = 3.0-6.8). The OR for vulvar cancer was 18.8 (95% CI = 11.9-29.8) among current smokers who were HPV16 seropositive in comparison with never smokers who were HPV16 seronegative. CONCLUSIONS: Current smoking, infection with HPV16, and infection with HSV2 are risk factors for vulvar cancer. Risk appears particularly strong among women who are both current smokers and HPV16 seropositive.

Efficient oncolysis by a replicating adenovirus (ad) in vivo is critically dependent on tumor expression of primary ad receptors.

Replicating adenoviruses (Ads) are designed to replicate in and destroy cancer cells, generating viral progeny that spread within the tumor. To address the importance of the primary cellular receptor for Ads, the coxsackievirus and Ad receptor (CAR), in permitting intratumoral spread of a replicating Ad, we have used a pair of tumor cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the first direct evaluation of the relationship between the efficacy of a replicating Ad and the primary receptor levels of the host cell without the confounding influence of other variable cellular factors. We demonstrate that the absence of the primary cellular receptor on the tumor cells restricts the oncolytic potency of a replicating Ad both in vitro and in vivo. Based on these findings, it is apparent that the potential therapeutic advantages afforded by viral replication would be negated by poor intratumoral spread of the viral progeny due to the failure to infect neighboring tumor cells. Because a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect Ads to achieve CAR-independent infection will be necessary to realize the full potential of replicating Ads in the clinical setting.

Structure of the lipid-containing bacteriophage phi 6. Disruption by Triton X-100 treatment.

The structure of the lipid-containing bacteriophage phi 6 was studied by means of controlled Triton X-100 disruption and subsequent isolation of subviral particles. Rate-zonal centrifugation yielded two fractions, a nucleocapsid fraction with RNA, proteins P1, P2, P4, P7, P8, and about half of the protein P5 and a membrane fraction with associated proteins P3, P6, P9, P10, and the rest of the protein P5. Following isopycnic sucrose gradient centrifugation, an empty capsid fraction was obtained which lacked RNA but contained a protein composition similar to the nucleocapsid except for the absence of P5. The membrane fraction isolated after isopycnic centrifugation was morphologically indistinguishable from that isolated after rate-zonal centrifugation but contained only proteins P3, P6, P9 and P10. By treating phi 6 with Triton X-100 prior to isopycnic sucrose gradient centrifugation the viral membrane was further separated into submembrane structures and the attachment protein, P3, could be isolated in rather pure form.

Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy.

BACKGROUND: There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS: Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS: Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Arrangement of double-stranded DNA packaged in bacteriophage capsids. An alternative model.

Toroidal winding of double-stranded DNA in the protein capsids of bacteriophages has been proposed previously. An alternative model for the packaging and arrangement of DNA in bacteriophage capsids is presented here. By introducing sharp folds, the alternative model avoids toroidal winding and its accompanying difficulties. This alternative model is in agreement with the current data obtained with several different bacteriophages.

The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II.

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.

Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.

The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.

Location of filamentous phage minor coat proteins in phage and in infected cells.

We show that all minor coat proteins of phage f1 are integral inner membrane proteins prior to assembly. Hence all phage structural and morphogenetic proteins are localized in the membrane of the infected cell, consistent with models of phage assembly in which morphogenesis is coincident with phage extrusion. Our data suggest that the minor coat proteins, pVI and pIII, are already associated with the major coat protein, pVIII, in the membrane. On the other hand pVI and pIII are not recovered as a complex from the membrane, even though experiments with dissociated phage show they are associated in phage. The minor coat proteins, pVII and pVIII, are also associated in phage. With the use of sera directed against the minor proteins, we show that the minor coat protein, pIX, is accessible in intact phage, but pVI and pVII are not. Consistent with earlier results, the attachment protein, pIII, clearly is exposed on the phage exterior. Infected cells contain about ten times more pVII and pIX than is incorporated into phage.

Genetic divergence among the group B coxsackieviruses.

As documented in the preceding discussion, the noncoding regions, and in particular the 5' NTR, of the CVB are tolerant of a substantial degree of nucleotide diversity while still being capable of fulfilling the life cycle requirements for these viruses. While diversity among the CVB is observed in the sequences encoding for the capsid proteins, it tends to involve predominantly those regions coding for amino acids located at the surface of the virus and not those responsible for the structural integrity of the mature virion, i.e., beta-barrels and alpha-helices. It is these capsid surface differences that define the six serotypes of the CVB and subdivide them antigenically into strains. Additionally, these proteins most likely play the major role in determining host and cellular tropism. The most conserved of the CVB proteins and, therefore those with the least diversity in their coding sequences, appear to be the nonstructural proteins. Perhaps, as speculated earlier, it is a conformational requirement imposed by the necessity to interact with host or viral substrates that maintains the high degree of amino acid identity of this group of viral proteins.

Osteoclasts in human osteopetrosis contain viral-nucleocapsid-like nuclear inclusions.

We report the discovery of nuclear inclusions in the osteoclasts of three unrelated patients with benign osteopetrosis that resemble the osteoclast inclusions characteristic of Paget's disease of bone. These inclusions are morphologically and dimensionally identical to the nucleocapsids of a virus of the Paramyxoviridae family. Supporting a possible viral association with benign osteopetrosis in the observation of the presence of antigens of respiratory syncytial virus, measles virus, and/or mumps virus in the cells of all five patients whose paraffin-embedded bone specimens were tested. These included two patients whose osteoclasts contained nuclear inclusions. No patients with the malignant form of the disease have been studied. There is as yet no proof that a virus is causally related to human osteopetrosis even though a virus can produce an avian form of the disease.

Role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract.

To evaluate the hypothesis that innate immune mechanisms play a major role in eliminating adenovirus (Ad) vectors from the lung, the fate of adenoviral genome of an Ad vector was quantified in the first 24 h after intratracheal administration of an Ad vector coding for beta-galactosidase (beta gal) to mice. Southern analysis with an Ad specific probe showed that 70% of the Ad genome was lost within 24 h, in both immunocompetent and immunodeficient animals. When alveolar macrophages were eliminated by administration of liposomes containing dichloromethylene-biphosphanate, subsequent administration of Ad vector was associated with a 100%+/-8% increase in lung Ad DNA and 96%+/-9% rise in beta gal expression at 24 h compared to control animals. In vitro infection of mouse, rat, and human alveolar macrophages with an Ad vector resulted in 65% loss of vector genome within 24 h, whereas the vector genome was stable in lung epithelial cell lines. PCR in situ hybridization demonstrated that the Ad vector genome persisted A549 lung epithelial cell in vitro but not in alveolar macrophages. Finally, alveolar macrophages recovered from the mouse lung 30 min following intratracheal administration of an Ad vector showed large amounts of vector genome, whereas much less was evident in alveolar macrophages recovered after 24 h. These observations demonstrate that alveolar macrophages play an important role in elimination of Ad vectors from the lung and suggest that strategies to transiently suppress this major innate immune defense system might be rewarding in enhancing the efficiency Ad vectors for lung gene therapy.

Virus particles of cucumber green mottle mosaic tobamovirus move systemically in the phloem of infected cucumber plants.

Systemic movement through the phloem of infected host plants is a key process in the life cycle of plant viruses, knowledge of which is scant. A main point to be elucidated is the structural form in which virus infection moves within the phloem. Indirect evidence suggests that virions might be the viral structure that moves in the phloem, but data from direct analysis in phloem sap have not been reported. We have done such analysis in the system cucumber (from which phloem exudate can be collected)/cucumber green mottle mosaic tobamovirus (CGMMV). CGMMV has structurally well-characterized particles. Both CGMMV coat protein and RNA were found in phloem exudate from infected cucumbers. Analysis of the accessibility of CGMMV RNA in phloem exudate to RNase A indicates that it is protected within a ribonucleoprotein structure. The accessibility to RNase A of the RNA in these structures was as in virus particles. Centrifugation analyses showed that the ribonucleoprotein structures in the phloem exudate have the same mass and isopycnic density as virions. Virus particles indistinguishable from purified virions were detected by electron microscopy in phloem exudate. No evidence of free RNA or other CGMMV-related structure was found in phloem exudate of infected plants. These results indicate that CGMMV movement in the phloem occurs mainly, if not exclusively, in the form of virus particles.

Demonstration of in vitro synthesis of human papilloma viral proteins from hand and foot warts.

HPV particles purified from [35S]-methionine labeled and unlabeled halves of single hand and foot warts have been fractionated into empty, light full, and heavy full particles by buoyant density gradient centrifugation, and their proteins analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE) and visualized by either fluorography or silver staining. The L1 coat protein (54 Kd) was found in trace amounts in unmodified and slightly modified forms in the labeled empty and light full particles but could not be detected in the labeled heavy particles. L1 appeared to exist in the three unlabeled particle types in differentially modified forms. A putative L2 protein was also found to be modified (74-80 Kd) and was found preferentially in the unlabeled heavy full particles. The commercial cross-reactive BPV antibody recognized a labeled 58-Kd protein found predominantly in the empty and light full particles and a pair of proteins (41-42 Kd) found unlabeled in the heavy full particles. Besides L1, there were several other proteins (IEF 40 Kd; NEPHGE 42, 38, and 36 Kd) which were detected labeled in the empty particles and in increasing unlabeled amounts in the light full and heavy full particles. Four proteins (IEF 66, 13 and 11 Kd, and NEPHGE 9 Kd) were found exclusively in the full particles and may be involved in packing the viral genome. These observations suggest that a virus particle assembly pathway exists from the empty particles, via the light full, to the mature heavy full particles.