Immature Core Research Articles

Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates mainly with viral particles containing unprocessed gag.

Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.

Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

Generation of infectious virus particles by transient co-expression of human immunodeficiency virus type 1 gag mutants.

We have demonstrated that COS7 cells transiently co-expressing myristylation-defective (Myr-) and protease-defective (PR-) human immunodeficiency virus (HIV) mutants can release infectious virions when co-transfected with an amphotropic murine leukaemia virus envelope protein expression plasmid (SV-A-MLV-env). In contrast, no infectious virions were detected when a PR-, noninfectious HIV gag mutant was co-expressed with the Myr- mutant, although the Myr- mutant could still process the immature core particles in trans. This result indicates that generation of functionally normal Gag proteins is required for virus infectivity in our complementation system. A mutant with a 56-amino-acid deletion in the N-terminal region of the capsid (CA) domain could still complement the PR- mutant to generate infectious virions, suggesting that the deletion mutant could provide a functional protease for processing in the PR- mutant. This result is consistent with the concept that mutations within the N-terminal region of the CA domain have no major effects on Gag-Pol incorporation into particles.

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.

Hydrous pyrolysis of a Type III source: fractionation effects during primary migration in natural and artificially matured samples

A core from the Cambay Shale Formation of the Cambay Basin, containing immature Type III organic matter, was pyrolysed at 300°C for different durations of time to different maturation levels. Fractionation effects were studied employing a three-step extraction technique after removal of the expelled pyrolysate. The extractable organic matter (EOM) obtained on extraction of the whole core is assumed to be that present in open pores, while that obtained on finely crushing the sample is assumed to be that present in closed pores. The EOM obtained from 1 cm chips is termed EOM from semi-open pores. The gross composition of the pyrolysates expelled during pyrolysis is not similar to the oils reservoired in the area, and there is no significant fractionation observed between expelled pyrolysates and unexpelled EOM. Our study indicates movement of fluids between closed, semi-open and open pores. In both systems, there is a higher concentration of EOM in open pores than in semi-open and closed pores, and the fraction of EOM in open pores is much greater in the artificial system than in the natural system. Fractionation effects on n-alkane and isoprenoid hydrocarbon-based parameters were also studied. n-Alkenes are present in semi-open and closed pores of the immature core and in the core after it was pyrolysed to 300°C for 6 and 48 h, but are absent in the open pores. n-Alkenes are present in closed pores in the naturally matured core. Presence of n-alkenes in the pyrolysates expelled during the 6 and 48 h experiments, but their absence in the open pores of the core, indicates that expulsion also occurs through temporary microfractures during laboratory pyrolysis, whereas in the natural system expulsion from closed pores seems to be only via semi-open and open pores.

The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity.

In vitro experimental infection of primary human hepatocytes with hepatitis B virus

Background/Aims: Studies on the interaction of hepatitis B virus (HBV) with its host cell require a suitable tissue culture system. This study used primary adult hepatocytes from healthy human liver tissue to establish productive infection in vitro. Methods: Hepatocytes were inoculated overnight with HBV. Production of viral proteins was assessed by radioimmunoassay and by [35S]methionine labeling, and production of viral DNA was assessed by Southern blotting and endogenous polymerase assay. Results: Secretion of high levels of hepatitis B surface antigen (HBsAg) and low levels of hepatitis B virus e antigen (HBeAg) into the medium was detectable 6 days after infection and reached maximum values after 12 days. Metabolic labeling showed production of viral proteins to be a result of de novo synthesis. The appearance of single-stranded HBV DNA in the cytoplasm of infected cells, typically present in immature cores, indicated viral replication. HBV DNA containing particles possessing an active viral DNA polymerase could be immunoprecipitated from the medium 12 days after infection. An antiserum specific for the preS1 region of the viral envelope was capable to block infection. Presence of dimethyl sulfoxide in the medium greatly improved the yield of viral proteins. Conclusions: Primary adult human liver cells are competent for infection with HBV.

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four is mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40° with any of the four is mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.

Processing, assembly, and immunogenicity of human immunodeficiency virus core antigens expressed by recombinant vaccinia virus

Recombinant vaccinia viruses that contained regions of the gag-pol open reading frames of human immunodeficiency virus type 1 (HIV-1) were constructed. Cells infected with recombinants containing both gag and protease genes expressed and processed HIV gag antigens efficiently. Processing was much reduced in cells infected with recombinants containing only gag, but not the protease gene. However, significant amounts of p41 were produced by proteaseddefective recombinants. This protein was immunoreactive with p24-specific monoclonal antibodies and was produced in a truncated form by a recombinant containing a 3′ deletion in the pt 5 coding region of gag ORF. These results indicate that p41 could represent an alternative gag precursor with N-terminal sequences derived from p24 and C-terminal from pt 5. Ultrastructural analysis of recombinant-infected cells revealed that the gag antigens expressed were assembled into retrovirus-like particles and were secreted into culture medium. This assembly process was not dependent on HIV protease function, because immature core particles were produced by recombinants lacking HIV-1 protease functions. Immunization of mice and chimpanzees with vaccinia-HIV gag recombinant viruses generated both antibody and cell-mediated immune responses to HIV gag antigens. These recombinants are therefore useful not only for studying HIV virion processing and assembly, but also for designing immunogens for the prophylaxis and immuno-therapy against AIDS.

Regulation by dimethylsulfoxide, insulin, and corticosteroids of hepatitis b virus replication in a transfected human hepatoma cell line

A human hepatoblastoma cell clone E4 was obtained by transfection of HepG2 cells with a plasmid DNA containing four tandem copies of hepatitis B virus (HBV) genome. Analysis of both intracellular and extracellular viral DNA revealed that this clone exhibited the main steps of the replication process previously found in normal hepatocyte primary cultures experimentally infected in vitro. Indeed, relaxed-circular, covalently closed circular, and single-stranded forms of viral DNA were identified in the cells together with complete virions and immature cores in the medium. Furthermore, the ability of these secreted particles to infect normal human hepatocyte cultures was established. These E4 cells were used to evaluate the effect of various soluble factors on HBV replication. Corticosteroids and, to a greater extent, dimethylsulfoxide (DMSO) increased intracellular viral DNA, whereas insulin reduced it dramatically. Parallel changes in the amounts of viral DNA secreted in the medium were observed. Measurement of the albumin secretion rate indicated that cellular and viral activities could be regulated, at least in part, in a coordinated manner.

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor.

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.

Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.

Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores

Double immunofluorescence assays of fixed sections of tissues from Pekin ducks congenitally infected with duck hepatitis B virus were carried out to score cells reactive to antiserum elicited either to viral DNA synthesis complexes (immature cores) purified from liver of infected ducks or to viral surface antigen particles and virions purified from sera of viremic ducks. Subpopulations of doubly fluorescent cells were detectable in liver (hepatocytes and bile duct epithelia), in kidney (proximal tubular epithelia), and in pancreas (acinar-associated cells and endocrine α and β cells); all cells reactive with one antiserum were reactive with the second antiserum. Competition assays established that the cell-associated antigens recognized by the two antisera were mutually non-crossreactive. The cell-associated antigen recognized by the antiserum elicited to the purified immature cores appears to be crossreactive with a 35,000-Da polypeptide fraction associated with immature cores.

Separation of a murine leukaemia virus protein kinase activity from its Pr65gag polyprotein substrate after DNA--cellulose chromatography.

We have recently found that, in vitro, the murine leukaemia virus (MuLV)-associated protein kinase activity predominantly phosphorylates Pr65gag, a virus protein present in relatively small amounts in partially purified virus preparations. Other virus proteins, such as p10, Pr27gag and Pr40gag, are also phosphorylated in vitro, but to a lesser degree. Furthermore, when immature core subparticles which are enriched in Pr65gag are prepared from virions by Sepharose 6B exclusion column chromatography, about 50% of the kinase activity (as assayed with the exogenous substrate phosvitin) remains associated with the cores. We report here that this core-associated activity is distinct from Pr65gag since it can be separated from Pr65gag by chromatography on denatured DNA--cellulose columns followed by centrifugation of the 0.2 M-NaCl-eluted fraction. Under these conditions, Pr65gag is pelleted while the kinase activity, which can phosphorylate both endogenous (MuLV Pr65gag and p10) as well as exogenous (phosvitin) substrates, remains in the supernatant. Interestingly, when the amount of Pr65gag is reduced, as in such preparations, p10 then becomes more heavily phosphorylated.

In vitro phosphorylation of murine leukemia virus proteins: Specific phosphorylation of Pr65 gag, the precursor to the internal core antigen

In vitro phosphorylation of Nonidet P-40 disrupted murine leukemia virus with [γ- 32P]ATP showed that Pr65 gag, which is present as a relatively minor component (14% of total Coomassie blue-stained virus proteins), was the major (80% labeling) phosphorylated species. A small amount of 32P label (i.e., 3–9%) was associated with proteins whose mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the same as Pr40 gag, Pr27 gag, p12, and p10. In contrast, long-term (20 hr) in vivo phosphorylation with [ 32P]orthophosphate showed that p12 was the most highly phosphorylated viral protein species. In neither case did we find any label associated with the major virion proteins, p30, p15, or gp70. These results suggest that phosphorylation is specific for certain viral proteins and, further, that if Pr65 gag is packaged as an uncleaved polyprotein into virions it remains relatively unphosphorylated. We have also found that the kinase activity which phosphorylates Pr65 gag in vitro is tightly associated with immature core subparticles and can phosphorylate the exogenous substrate phosvitin. The same or an additional kinase activity can also be detected in the soluble fraction of detergent-disrupted virus using endogenous viral proteins or phosvitin as substrate. Based on an analysis of the tryptic peptide patterns of pPr65 gag phosphorylated in vitro and in vivo, it appears that the in vitro phosphorylation of Pr65 gag occurs at serine residues which are identical to or similar to those found for pPr65 gag in vivo, i.e., on pp12, as well as at another site. This other site is also probably on p12 since in vitro phosphorylated pPr65 gag when cleaved in vitro by the murine Pr65 gag proteolytic factor, produces as its initial product a phosphorylated vand that has the molecular weight of pPr27 gag and the antigenic determinants of p15 and p12.

A mutation suppressing the overproduction of RNA polymerase beta beta' subunits in the RpoC1 strain of Escherichia coli.

As has been shown previously, RNA polymerase β′ subunit ts-mutation rpoC1 results in an overproduction of RNA polymerase ββ′ subunits at nonpermissive temperature. The mutant enzyme shows low activity in vitro and a sedimentation coefficient 9S which is characteristic of immature core polymerase. In this paper we describe a mutation designated opr1 which suppresses RNA polymerase ββ′ subunit overproduction. The mutation was found among Ts+ revertants of the Ts double mutant carrying a rpoC1 mutation and a rif-r rpo B251 mutation. Opr1 is closely linked to the original rpo mutations and shows complete trans-dominance. Although opr1 seems to affect RNA polymerase, it does not suppress the accumulation of immature 9S RNA polymerase and does not restore the activity of the RpoC1 mutant enzyme. This and other results of a comparison of strains carrying different combinations of rpoC1, rpoB251 and opr1 mutations suggest that neither inhibition of total RNA and protein synthesis, nor the low RNA polymerase activity in vitro, nor the apparent defects in enzyme maturation, nor the enzyme degradation observed at 42°C are responsible for the ββ′ overproduction in RpoC1 strains.