Further studies on the hyperphosphorylated form (p40) of the rabies virus nominal phosphoprotein (P)

We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.     

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).     

Further studies on the soluble form (gs) of rabies virus glycoprotein (g): molecular structure of gs protein and possible mechanism of the shedding

In this study, we investigated the antigenic structures and maturation of some C-terminal-deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope-specific anti-G mAbs, but took the 1-30-44 epitope-deficient conformation (termed G(C) form). (The 1-30-44 epitope was acid-sensitive and dependent on two separate regions, the Lys-202-containing and Asn-336-containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507-519). Intact G proteins took the 1-30-44 epitope-positive form (referred to as G(B) form) on the cell surface, but not inside the cell. A deletion mutant G(1-429) (termed GDeltaTC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C-terminal-deficient mutant G(1-462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C-terminus, but was transported to the cell surface, where it showed pH-dependent cell fusion activity and almost full antigenicity to most of the anti-G mAbs with the exception of very weak antigenicity to mAb #1-30-44. No Gs protein could be detected in the CT1-producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1-30-44 epitope-positive G(B) conformation. Gs proteins might have lost the C-terminal regions during the maturation process after being exported from the rER.     

Association of rabies virus nominal phosphoprotein (P) with viral nucleocapsid (NC) is enhanced by phosphorylation of the viral nucleoprotein (N)

We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase-treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation-deficient NC-like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PDeltaN19, which lacked the N-terminal 19 amino acids and was capable of binding to the NC-like structures but incapable of forming the RNA-free N-P complexes. Immunoprecipitation studies with mAb #402-13 further suggested that the NC-specific linear 402-13 epitope was exposed even on the P proteins which were associated with the phosphorylation-deficient NC-like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC-like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402-13 epitope-positive P protein and the NC probably by stabilizing such P-NC binding.     

我国四株狂犬病毒M和P基因序列分析和所编码蛋白的分析

为了解我国狂犬病毒M、P基因序列和结构特点,用RT-PCR方法获得目的基因片段,测定核苷酸序列后,计算机分析核苷酸和氨基酸序列及其功能区位点结构。结果显示四株病毒M基因核苷酸和氨基酸序列同源性分别为83.9%～99.5%和93.1%～99%,四株狂犬病毒M蛋白上调节病毒RNA转录和复制功能的第58位氨基酸残基均为谷氨酰胺残基(E),与特异性细胞蛋白WW区域作用的PPxY结构序列均为PPEY保守序列;四株病毒P基因核苷酸和氨基酸序列同源性分别为83.6%～99.8%和87.2%～99%,P蛋白与胞浆动力蛋白轻链LC8相互作用的序列位于143～148位氨基酸残基,均为DKSTQT,四株病毒P基因与L蛋白、N蛋白作用位点序列显示未发生影响其生物学功能的变异。研究结果证实了这两种蛋白结构在病毒致病性中起重要作用的推论。     

Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.     

Characterization of a slow-migrating component of the rabies virus matrix protein strongly associated with the viral glycoprotein

We investigated multiple forms of rabies virus matrix (M) protein. Under non-reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23- and 24-kDa) of M protein, an M antigen-positive slow-migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54-kDa and monomer components in the virion were about 20-30% and 70-80% of the whole M protein, respectively, while the content of the 54-kDa component was smaller (about 10-20% of the total M protein) in the cell than in the virion. The 54-kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% nonionic detergents by which most monomer components were solubilized. The 54-kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3-9-16), which recognized a linear epitope located at the N-terminal of the M protein. The mAb #3-9-16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54-kDa component of the M protein. Monomers and the 54-kDa polypeptide migrated to the same isoelectric point (pI) in twodimensional (2-D) gel electrophoresis, implicating that the 54-kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen-positive 54-kDa polypeptide is a homodimer of M protein, taking an N-terminal-exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.     

Two different conformations of rabies virus glycoprotein taken under neutral pH conditions

We previously reported that the rabies virus glycoprotein (G) takes either of two different conformations (referred to as B and C forms) under neutral pH conditions, that could be differentiated by their reactivity to a monoclonal antibody (mAb), #1-30-44, that recognizes the acid-sensitive conformational epitope, and the formation taken is dependent on two separate regions containing Lys-202 and Asn-336 of the protein (Kankanamge et al., Microbiol. Immunol., 47, 507-519, 2003). Semi-quantitative antibody-binding assays demonstrated that only one-third to one-fourth of mature G proteins on the cell surface were taking the 1-30-44 epitope-positive B form even at pH 7.4. The ratio of B to C varied, depending on the environmental pH, but did not decrease to zero even at pH 5.8-6.2, preserving a certain content (about 15-20%) of B form. Immunoprecipitation studies demonstrated that a portion of G proteins were intimately associated with a dimer form of matrix (M) protein in terms of resistance to treatment with a mixture of 1% deoxycholate and 1% Nonidet P-40, and seemed to preserve the B form even at lower pHs. Similar results were also obtained with the virion-associated G proteins, including the intimate association of a portion of the G proteins with the M protein dimer. From these results, we assume that a certain portion of the rabies virion-associated G proteins are associated with a dimer form of M protein, keeping the 1-30-44 epitope-positive B conformation under various pH conditions, which might possibly assure the virion's recognition of host cell receptor molecules in the body.     

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58^IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58^IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58^IPK-mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.     

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.     

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.     

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.     

Molecular cloning of the gene encoding an acidic ribosomal protein of the P2 family from the ciliate Euplotes raikovi

We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear "gene-sized" molecules with Euplotes telomeres at the ends. The longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. To our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

RNA-Binding Properties of the Proteins of Beet Yellows Closterovirus

Recombinant full-sized proteins p64, p65, p24, p22, p21 and pcp, hel, mtr, and pol fragments of the replicative polyprotein of beet yellows closterovirus were purified and tested for RNA binding. North-Western blotting revealed the RNA-binding activity for p64 and hel (a 21-kDa fragment of the helicase domain with conserved motifs V and VI). Gel retardation assay confirmed hel binding with a randomized RNA probe in vitro, and a cooperative RNA–hel interaction was assumed on evidence of the binding pattern. The RNA–hel complexes proved to be stable at a high ionic strength.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

Bovine P2 Protein: Sequence at the NH2-Terminal of the Protein

Sequence data from key fragments of the P2 protein established the order of cyanogen bromide (CNBr) peptides in the structure of the protein and the primary structure for approximately one-half of the molecule. Data were obtained from the three tryptic peptides of blocked NH2-terminal CNBr peptide (CN3), the large CNBr peptide of P2 protein (CN1), and a fragment obtained from P2 by cleavage at tryptophan with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. This last fragment was found to contain an over-lapping sequence that proved the juxtaposition of CN1 and CN3 in P2 protein. Thus, based on this fact and the characteristics of the CNBr peptides, the P2 structure is composed of CNBr peptides in the order: CN3-CN1-CN2(Val)-CN2(Lys). A comparison was made between the partial sequence of P2 protein and the equivalent portion of the structure of bovine myelin basic protein. The structures of these two proteins were found to be distinctly different although certain similarities are found.     

Properties of exogenously added GPI-anchored proteins following their incorporation into cells.

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.     

Characterization of 40,000- and 25,000-dalton intermediate precursors to Rauscher murine leukemia virus gag gene products.

Under steady-state labeling conditions, Rauscher murine leukemia virus-infected NIH Swiss mouse cells contain at least three major polyproteins derived from the viral gag gene. They have molecular weights of 65,000, 40,000, and 25,000. They have been termed pPr65gag, Pr40gag, and pPr25gag. pPr65gag has been shown by a number of laboratories to be composed of all four core proteins (p15, pp12, p30, and p10). In this paper, Pr40gag was found to contain p30 and p10 antigenic determinants and peptide sequences, whereas pPr25gag was found to contain p15 and pp12. Pr40gag and pPr25gag are rapidly labeled precursor proteins that were detectable early in pulse-chase experiments. Both precursors disappeared during the later stages of the chase period concurrent with the appearance of the mature viral core proteins. pPr65gag and pPr25gag were found to be phosphorylated, pPr25 having a higher specific activity of 32P than pPr65. In spite of this, peptide mapping studies, as well as the identification of the phosphorylated amino acid residues of pPr65, and pPr25, and pp12, indicated that the same sites are phosphorylated regardless of whether the precursors or the mature pp12 are examined.