Specific in vitro association between the hepatitis C viral genome and core protein.

Little is known about the molecular interactions required for hepatitis C virion assembly. The 5' noncoding region (5'NCR) of the RNA genome is highly conserved and has extensive secondary structure. The highly basic core protein is rich in arginine and lysine residues. We postulate that a specific interaction between these structures may be important for virion assembly. Using an RNA gel mobility shift assay, a specific interaction has been demonstrated between the RNA of the 5'NCR and recombinant core protein. Proteins from other regions of the virus do not interact with the viral RNA. The interaction is inhibited competitively by unlabelled sense polarity RNA, but antisense 5'NCR RNA and nonspecific RNAs compete only at much higher concentrations. These data suggest that there is a specific interaction between the 5'NCR of the hepatitis C virus (HCV) genome and HCV core protein. This interaction may be important for the specific encapsidation of the viral genome during HCV replication.

Presence of viral replicative intermediates in the liver and serum of patients infected with hepatitis C virus.

Hepatitis C virus (HCV) is a positive-polarity, single-stranded RNA virus, distantly related to the pestivirus and flavivirus genera. These viruses replicate through the formation of a minus-strand RNA intermediate, which encodes the positive-strand genome, which is subsequently encapsidated, enveloped, and released from infected cells. Minus-strand RNA is not found in the mature, circulating virions of flaviviruses. In an attempt to study the relative amounts of viral plus and minus strand in the liver and serum of HCV-infected individuals, we have developed a technique to amplify specifically each of the viral strands using a modified reverse transcriptase/polymerase chain reaction protocol on extracted RNA. Liver tumor and nontumor tissue from a patient with C-100-3 antibody was analyzed using this technique. In both cases, viral plus and minus strands were detected, although the plus-strand signal was several fold stronger than minus-strand signal by Southern hybridization. Sera from 11 C-100-3 antibody-positive patients with abnormal serum AIT levels were similarly analyzed. In all cases viral plus strand was detected, and in 10 of 11 cases viral minus strand was detected. The minus-strand signal was always much weaker than the plus-strand signal and the ratio of plus strand to minus strand varied among patients. No correlation was found between the level of minus strand detected or its ratio to plus strand with the level of serum transaminases or any other clinical parameter.

Activation of protooncogene c-jun by the X protein of hepatitis B virus.

A specific viral oncogenic mechanism has not been shown for hepatitis B virus (HBV), although persistent HBV infection has been strongly associated with the development of hepatocellular carcinoma (HCC). Most HCCs in HBV carriers contain integrated viral sequences in host DNA and this raises the question of whether such integrations ever contribute to oncogenesis. HBV does contain a gene (designated the hbx gene) which encodes a transcriptional trans-activator protein capable of activating homologous and heterologous regulatory sequences. Hbx has been detected in some human HCC with HBV integrations and the expressed hbx protein appears to have transcriptional transactivating activity. These findings raise the possibility that hbx expression could contribute to hepatocarcinogenesis by activating cellular genes that could contribute to oncogenicity. The possibility that the hbx protein may activate certain protooncogenes was investigated and we found that hbx can activate the protooncogene c-jun promoter. c-Jun was found to be expressed at a very low level in normal liver tissue but at high levels in HCCs of HBV-infected patients.

Transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat by hepatitis B virus X-protein requires de novo protein synthesis.

Human hepatitis B virus (HBV) X-gene, previously shown to be capable of trans-activating heterologous regulatory elements of the human beta-interferon gene, the human immunodeficiency virus type I (HIV-1) long terminal repeat (LTR), the simian virus 40 (SV40), and HBV, has the capacity to code for a 17-kDa polypeptide (designated pX17). We now report that pX17 synthesized in Escherichia coli can activate transcription controlled by the HIV-1 LTR using a protoplast fusion technique. Protoplasts of E. coli-containing presynthesized X-protein were fused with lymphocytic H938 cells harboring an integrated copy of a plasmid with the CAT gene under control of the HIV-1 LTR (HIV-1 LTR CAT) and a marked increase in the steady state expression of the CAT mRNA was observed. When the same fused cells were treated with the protein synthesis inhibitor cyclohexamide, the pX17-dependent activation of the HIV-1 LTR was abolished. This result indicates that the X-protein expressed in E. coli is biologically active and suggests that the HBV X-protein-mediated trans-activation of the HIV-1 LTR in this system requires de novo cellular protein synthesis.

Hepatitis B virus X gene activates kappa B-like enhancer sequences in the long terminal repeat of human immunodeficiency virus 1.

The role of the hepatitis B virus (HBV) X gene during virus infection has not been defined. We previously showed that expression of the HBV X gene in the human hepatocellular carcinoma cell line HepG2 trans-activates chloramphenicol acetyltransferase gene expression under control of the human immunodeficiency virus 1 (HIV-1) long terminal repeat and we have now identified a specific sequence in the HIV-1 long terminal repeat that is responsive to the HBV X gene. Plasmid constructs with the chloramphenicol acetyltransferase gene regulated by an isolated and twice-repeated 12-base-pair HIV-1 enhancer sequence homologous to the nucleotide sequence that binds the nuclear transcription factor NF-kappa B (the HIV-1 kappa B-like sequence) were trans-activated by the HBV X gene in HepG2 cells, indicating that the kappa B-like enhancer sequence in the HIV-1 long terminal repeat is responsive to the X gene. When eight copies of the HIV-1 kappa B-like sequence were used to regulate beta-globin gene expression, transcription of this gene was activated by the HBV X gene in HepG2 cells and no beta-globin gene transcription was detected in the absence of the HBV X gene. beta-globin gene expression regulated by the activator protein 2 (AP-2) binding sequence was not activated by the HBV X gene. Treatment of HepG2 cells with phorbol ester resulted in modest activation of the HIV-1 kappa B-like enhancer sequence suggesting that an NF-kappa B-like factor was induced in these cells as it is in T lymphocytes by phorbol ester; however, phorbol ester did not demonstrably enhance the activation of the HIV-1 enhancer observed with the HBV X gene. These experiments indicate that the HIV-1 kappa B-like transcriptional enhancer sequence is activated by the HBV X gene and suggest that the HBV X gene might play a role in regulating transcription of a gene under control of a kappa B-like enhancer during HBV infection. Since such a sequence has not been found in the HBV genome and HBV gene expression appears not to be regulated by the HBV X gene, a cellular gene that plays a role in HBV replication could be the target of the X gene during HBV infection.

Transcription of the human beta interferon gene is inhibited by hepatitis B virus.

The manner by which the trans-acting factor encoded by the 1,828-base-pair (bp) BamHI DNA fragment of hepatitis B virus (HBV) suppresses the production of human beta interferon was determined. Steady-state levels of RNA specific for human beta interferon were decreased in cells that contained the 1,828-bp BamHI DNA fragment of HBV. The reduced accumulation of interferon-specific RNA was due to an inhibition of transcription of the interferon gene by the HBV trans-acting moiety. The expression of the interferon gene that is under the control of a heterologous promoter such as the simian virus 40 early promoter was not altered by the presence of the 1,828-bp BamHI HBV DNA fragment. In contrast, the HBV moiety inhibited the expression of the cat gene, whose expression is controlled by the regulatory DNA region of the human beta interferon gene. These results indicate that the HBV trans-acting moiety suppresses the expression of the human beta interferon gene at the transcriptional level by interacting with the regulatory DNA sequences 5' to the coding sequences for beta interferon.

Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X.

Hepatitis B virus (HBV) X-gene product activates transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). To identify a cis-acting regulatory sequence within the HIV-1 LTR which is responsive to the HBV X-gene trans-activating function, we examined the effects of HBV X-gene expression in cells with a series of LTR/CAT deletion mutants. A region of the HIV-1 LTR containing the previously identified kappa B-like enhancer element was found to be responsive to HBV X-gene activation, and this effect was independent of, and additive with, the effect of the HIV-1 tat-III protein on CAT expression. Since kappa B-like enhancer sequences are known to regulate transcription of a variety of viruses and cellular genes, our results suggest that the X gene could activate such a gene during HBV infection and replication.

Hepatitis B virus X gene can transactivate heterologous viral sequences.

The smallest open reading frame of hepatitis B virus (HBV) has been designated the X gene and its biological function during HBV infection and replication is not known. Experiments described here demonstrate that expression of the HBV X gene in HepG2 cells containing a plasmid with the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) sequence leads to a marked increase in CAT gene transcription as well as expression of the gene product (CAT). The HIV-1 tatIII gene and the HBV X gene together increased HIV-1 LTR-regulated CAT expression above that observed with either gene alone, suggesting a synergistic effect of the X gene and tat. HBV X gene also stimulated expression of the CAT gene under control of the simian virus 40 enhancer and early promoter but not the visna virus LTR or the human T-cell lymphotropic virus type I (HTLV-I) LTR, indicating that the HBV X gene can transactivate some but not other heterologous viral sequences. Transactivation of the HIV-1 LTR by the HBV X gene varied in different cell lines, suggesting that it may be mediated by a cellular factor(s).

Hepatitis B virus suppresses expression of human beta-interferon.

To determine whether hepatitis B virus (HBV) regulates the expression of the human beta-interferon gene, a series of recombinant bovine papilloma virus plasmids containing the human interferon gene and various fragments of the HBV genome were constructed and used to transform C127 cells, a murine fibroblast line. Analysis of the DNA from transformed C127 cells indicated that the interferon gene was intact and that the plasmids replicated as stable multicopy elements. The 1828-base-pair BamHI HBV DNA fragment containing the core antigen gene, but not the 2755-base-pair Bgl II HBV DNA fragment encoding both the surface antigen and the X antigen, suppressed the production of human beta-interferon. No effect by any of the recombinant plasmids on the synthesis of murine interferon was detected. The suppression of human beta-interferon by HBV occurs via a trans-acting factor. A frameshift mutation within the HBV core gene alleviates the inhibitory activity; thus we infer that the core protein is this factor or is crucially associated with this activity.

Transcriptional trans-activating function of hepatitis B virus.

The ability of hepatitis B virus (HBV) to stimulate the expression of a cellular gene was investigated by using a transient-expression system. A plasmid in which the expression of the bacterial chloramphenicol acetyltransferase (cat) gene had been placed under the control of the DNA sequences that regulate the expression of the human beta-interferon gene was constructed. In Vero cells, cotransfection of the 2.7-kilobase BglII DNA fragment of HBV together with the test plasmid containing the cat gene resulted in stimulation of the expression of the cat gene. This HBV DNA fragment was specific in its trans-activation; no significant stimulation of CAT activity was observed in constructs when the promoter and enhancer elements were derived from the murine sarcoma viral long terminal repeat, Rous sarcoma virus, BK virus, or simian virus 40. Results of subcloning of the HBV DNA fragment indicate that the trans-activating function resides in a 944-base-pair EcoRV-BglII DNA fragment of the HBV genome that contains the X structural gene and its promoter element. Removal of the promoter from the X structural gene resulted in loss of the trans-activating function. A frameshift mutation within the X gene region also eliminated the trans-activating activity. These results suggest that the X antigen could play a role in HBV infections by activating the expression of cellular genes.

Modulation of lymphocyte motility by macrophages.

Motility of lymphocytes plays a significant role in their functions. Because macrophages frequently associate with lymphocytes in lymphoid tissues and inflammatory sites, they are likely to be important in regulating lymphocyte motility. In this study, we identified a chemokinetic activity in macrophage culture supernatants. Interestingly, this activity could be detected by the capillary migration assay but not by the more commonly used Boyden chamber chemotaxis assay. Colchicine, on the other hand, was chemokinetic for lymphocytes in the Boyden chamber chemotaxis assay but not in the capillary migration assay. Both these observations and previous studies on the morphology of motile lymphocytes on two-dimensional (2-D) surfaces (capillary migration assay) and in 3-D matrices (Boyden chamber chemotaxis assay) suggest that lymphocytes possess more than one motility mechanism--one for 2-D surfaces and one for 3-D matrices. We propose that the macrophage-derived chemokinetic activity described herein only affected the motility mechanism on 2-D surfaces. In addition, we also observed that the chemokinetic activity was produced by "resting" macrophages and could not be augmented by further activation. Finally, the effect was greatest on mature T cells. We propose that this factor plays an important role in facilitating cell interactions within lymphoid tissues and inflammatory sites.

Aging and lymphocyte cytoskeleton: age-related decline in the state of actin polymerization in T lymphocytes from Fischer F344 rats.

T cell functions are known to decline with age, but the underlying cause of the decline is unclear. Because of the importance of cytoskeletal elements in cellular functions, we examined the content and the state of polymerization of actin in lymphocytes from Fischer F344 rats of four different ages (6, 14, 23, and 31 mo). The cellular actin content was determined by a DNAase I inhibition assay. Our results indicate that the total actin content of spleen lymphocytes did not change significantly with age; however, polymeric actin content, particularly in T cells, decreased with age, which might be a result of the shift from the polymeric actin pool to the monomeric pool. Similar changes also occurred in B cells but to a lesser extent. We conclude that the state of polymerization of lymphocytes changed drastically with age, and that this might be an important factor in the age-related decline in the cellular functions of lymphocytes.

Analysis of Tn3 sequences required for transposition and immunity.

Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity.

A role for hepatic lipase in chylomicron and high density lipoprotein phospholipid metabolism.

The rate of removal of phosphatidylethanolamine and phosphatidylcholine from the plasma of rats treated with antiserum to hepatic lipase was measured. The hepatic lipase antiserum was injected intravenously into animals prior to injection of 32P-labeled chylomicrons or 32P-labeled high density lipoproteins. In experiments in which 32P-labeled chylomicrons were injected, antiserum treatment inhibited removal of [32P]phosphatidylethanolamine from chylomicrons, and the unlabeled serum phosphatidylethanolamine levels increased 2-2.5-fold in 30 min. In contrast, hepatic lipase antiserum had no significant effect on the clearance of chylomicron [32P]phosphatidylcholine or on unlabeled phosphatidylcholine concentrations in serum after injection of chylomicrons. In experiments in which 32P-labeled high density lipoproteins were injected, the inhibitory effect of the antiserum on the rapid removal of [32P]phosphatidylethanolamine from the circulation was even more marked than its effect on the removal from chylomicrons. The removal of high density lipoprotein phosphatidylcholine on the other hand was unaffected by the antiserum, although a moderate increase in serum phosphatidylcholine concentration was seen. In antiserum-treated rats injected with 32P-labeled chylomicrons or high density lipoproteins, hepatic [32P]phosphatidylethanolamine radioactivity was decreased. Significantly more [32P]phosphatidylethanolamine was recovered from blood plus liver in the antiserum-treated rats, indicating that the antiserum inhibited the overall degradation of injected [32P]phosphatidylethanolamine. The data suggest that phosphatidylethanolamine is a preferred substrate for hepatic lipase in the metabolism of chylomicron and high density lipoprotein phospholipid.

Hepatic lipase. Purification and characterization.

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.

Mechanism of the age-related decline in lymphocyte proliferation: role of IL-2 production and protein synthesis.

Although an age-related decline in mitogen-induced proliferation in spleen lymphocytes has been reported by numerous investigators, the molecular mechanism responsible is unknown. In this study, we compared the mitogen-induced proliferation, IL-2 production, and protein synthesis in spleen lymphocytes isolated from 4, 12, 20, and 30 month-old male Fischer F344 rats. IL-2 production by Con A-stimulated lymphocytes, as determined by the ability of the culture supernatants to support the growth of cultured T cells, declined over 72% between 4 and 30 months of age. This decline in IL-2 production paralleled a similar decrease in proliferation. Early protein synthesis by Con A-stimulated spleen lymphocytes was determined by measuring the incorporation of [3H]-valine into acid insoluble material, and this dropped 74% between 4 and 30 months of age. There was a strong correlation between the age-related decline in the three parameters tested. Based on these results, we propose that the age-related decline in protein synthesis may be the molecular basis for the similar decrease in IL-2 production and mitogenesis.

Lipoprotein lipase activity of adult rat cardiocytes.

Cardiocytes were prepared by enzymatic dissociation of adult rat ventricular tissue, and comparative studies on lipoprotein lipase activity were conducted on fresh homogenates and acetone powders of these cells. Lipolytic activity in fresh homogenates was largely dependent on addition of serum activator to the assay, and the activity was sensitive to 1 M NaCl. Lipoprotein lipase activity was maximized in acetone powder preparations of cardiocytes. Approx. 10% of the total lipolytic activity was extractable from acetone powders of cells homogenized in the absence of serum, while approx. 50% was soluble from powders of cells homogenized with 10% serum. The non-extractable lipolytic activity was inhibited 80% with 1 M NaCl and about 47% with antibodies (IgG) to heart lipoprotein lipase. The buffer-extracted enzyme was completely sensitive to NaCl and was inhibited 80% by low concentrations of anti-lipoprotein lipase antibodies.

Regulation of lipoprotein lipase immunological study of adipose tissue.

An antibody to purified rat heart lipoprotein lipase was used to determine the relative specific activities of adipose tissue lipoprotein lipase from fed and fasted rats. The antibody was immobilized by coupling it to a Sepharose gel. This antibody bound approx. 80% of the lipoprotein lipase activity of extracts of rat adipose tissue. When the extracts were separated by gel chromatography into two lipase activity fractions (lipoprotein lipase "a" and lipoprotein lipase "b") and these fractions incubated with the antibody, only 10% of the lipoprotein lipase "a" activity was bound by the highest antibody concentration employed, whereas 93% of the lipoprotein lipase "b" was bound by the same amount of antibody. Increasing amounts of antibody incubated with extracts of adipose tissue of fed or fasted rats yielded similar titration curves. When a constant amount of antibody was incubated with increasing amounts of the adipose extracts, no significant difference was noted between extracts from fed and fasted animals. The data indicate that the high lipoprotein lipase activity of adipose tissue of fed rats, compared with that of rats fasted overnight, results from the presence of more lipoprotein lipase protein.

Antibodies to lipoprotein lipase. Application to perfused heart.

An antibody was prepared against purified rat heart lipoprotein lipase. 1. This antibody showed marked species specificity. It inhibited almost totally the lipoprotein lipase activity from all rat tissues examined (i.e., heart, adipose, postheparin plasma, and mammary gland), while having no effect on the activity of lipoprotein lipase partially purified from rabbit, guinea pig and bovine heart and from bovine milk. The antibody also had no effect on the hepatic lipase activity of rat postheparin plasma. 2. After antibody to rat heart lipoprotein lipase was recirculated for 5 min through isolated rat hearts, little or no lipoprotein lipase activity could be detected in the perfusate during 0-20 s of a subsequent non-recirculating perfusion with buffer containing 1 unit heparin/ml. 3. Following recirculation of antibody to lipoprotein lipase for 10 min and a non-recirculating perfusion with buffer for 2 min, the hearts no longer oxidized any significant amounts of 14C-labelled palmitate chylomicron triacylglycerol fatty acid to 14CO2 during a 15-min perfusion. The data give compelling evidence that the functional fraction of lipoprotein lipase in hearts is at the endothelial cell surface accessible to lipoprotein lipase antibody.