The testicular antiviral defense system: localization, expression, and regulation of 2'5' oligoadenylate synthetase, double-stranded RNA-activated protein kinase, and Mx proteins in the rat seminiferous tubule.

Although the involvement of viruses in alterations of testicular function and in sexually transmitted diseases is well known, paradoxically, the testicular antiviral defense system has virtually not been studied. The well known antiviral activity of interferons (IFNs) occurs via the action of several IFN-induced proteins, among which the 2'5' oligoadenylate synthetase (2'5' A synthetase), the double-stranded RNA-activated protein kinase (PKR), and the Mx proteins are the best known. To explore the antiviral capacity of the testis and to study the testicular action of IFNs, we looked for the presence and regulation of these three proteins in isolated seminiferous tubule cells, cultured in the presence or in the absence of IFN alpha, IFN gamma, or Sendai virus. In all conditions tested, the meiotic pachytene spermatocytes and the post-meiotic early spermatids lacked 2'5' A synthetase, PKR, and Mx mRNAs and proteins. In contrast, Sertoli cells constitutively expressed these mRNAs and proteins, and their levels were greatly increased after IFN alpha or Sendai virus exposure. While peritubular cells were also able to markedly express 2'5' A synthetase, PKR, and Mx mRNA and proteins after IFN alpha or viral exposure, only PKR was constitutively present in these cells. Interestingly, IFN gamma had no effect on peritubular cells' 2'5' A synthetase and Mx production but it enhanced Mx proteins in Sertoli cells. In conclusion, this study reveals that the seminiferous tubules are particularly well equipped to react to a virus attack. The fact that the two key tubular elements of the blood-testis barrier, namely, Sertoli and peritubular cells, were found to assume this protection allows the extension of the concept of blood-testis barrier to the testicular antiviral defense.

Purification of an atypical bovine interferon induced in response to heat shock in bovine cultured cells. Characterization in comparison with the classical alpha, beta and gamma interferon.

Heat-shock induced factor (HSIF) was excreted by bovine MDBK cells during their recovery period after a heat shock. This factor has the capacity to induce 2',5' oligoadenylate synthetase activity, an enzyme generally by interferon treatment (J Biol Chem (1987) 262, 4806-4811). We have observed that an antiviral state was also produced in response to heat shock. HSIF was purified 10,000-fold and different techniques showed a copurification of both activities. Certain properties of HSIF were established, such as its molecular mass (45 kDa) and isoelectric point (6.8). This cytokine was acid-sensitive as IFN gamma (type II) and temperature labile contrarily to alpha, beta and gamma bovine IFN. Immunoprecipitation and comparative chromatography on lectines or polynucleotides established that HSIF was structurally different from the three classes of bovine IFN. Moreover, two-dimensional electrophoresis and comparative analysis of [35S] methionine-labeled proteins induced by HSIF alpha, beta or gamma bovine IFN showed that HSIF induces a specific set of proteins. Taken together, all these results strongly suggest that HSIF is a new atypical bovine interferon induced in response to heat shock.

Antiviral activity of recombinant interferon-alpha on hepatitis A virus replication in human liver cells.

Human recombinant interferon-alpha (IFN-alpha) was assayed for its antiviral effect on hepatitis A virus (HAV) replication in the human hepatoma cell line PLC/PRF/5. IFN-alpha resulted in concentration-dependent reduction of HAV antigen expression and HAV replication. IFN-alpha had a prophylactic effect, but was still effective when it was added after the infection, even at the end of the first replication cycle. An important increase in 2',5'-oligoadenylate synthetase activity in the IFN-treated human liver cells was observed. The antiviral effect of IFN-alpha could be attributed to the induction of this enzyme. Moreover we have shown that IFN-alpha and glycyrrhizin were synergistic in their antiviral actions against HAV. IFN-alpha emerged, from the present study, as a promising candidate for chemotherapy of severe forms of hepatitis A.

Clinical value of the determination of an interferon-induced enzyme activity: studies of the 2'5' oligoadenylate synthetase activity in peripheral blood lymphocytes of patients.

The 2'5' oligoadenylate synthetase (2'5' A synthetase) is one of the interferon-induced enzymes. The measurement of its activity may thus reveal the presence of interferon, which is one of the body's non-specific antiviral, antitumor, and immunoregulatory agents. We found a constant level of this enzyme activity (mean value: 0.31 units +/- 0.13 S. D.) when measured in the white blood cells of healthy subjects (104). The majority of the patients with viral (27/30), bacterial (13/16) and autoimmune (15/16) diseases showed a 2'5' A synthetase activity greater than 0.57 units (mean value of the control + S. D.). Conversely the 2'5' A synthetase activity level was normal or low in malignancies and in diseases depending on other aetiologies than those described above. Therefore this simple an rapid biochemical assay seems to be useful for clinical study of infectious and inflammatory diseases.

Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases EcoRI, HindIII, HpaI, and XbaI.

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for EcoRI, HindIII, HpaI, and XbaI. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 x 10(6) for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 x 10(6)-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.

Ethanol induces 2',5'-oligoadenylate synthetase and antiviral activities through interferon-beta production.

We demonstrate here that ethanol, in contrast to heat shock (Chousterman, S., Chelbi-Alix, M.K., and Thang, M.N. (1987) J. Biol. Chem. 262, 4806-4811), induces interferon (IFN) synthesis and its related activities in Madin-Darby bovine kidney (MDBK) cells. The induced IFN is secreted maximally at 6 h, whereas the induction of 2',5'-oligoadenylate synthetase mRNA peaks between 9 and 12 h and its activity at 15 h. The appearance of both 2',5'-oligoadenylate synthetase activity and the antiviral state upon ethanol treatment is prevented by anti-bovine recombinant IFN-beta antibodies. Bovine diarrhea virus infection-free MDBK cells cultured in medium supplemented with serum substitute also gave similar results, thus indicating that IFN synthesis induced by ethanol is not mediated by the activation of bovine diarrhea virus. Together, these results show that: 1) ethanol induces the 2',5'-oligoadenylate synthetase and antiviral activities through IFN-beta production; and 2) the IFN produced does not act directly from inside the cells, but has to be first secreted to bind to its receptor. In MDBK cells, ethanol induces the synthesis of the 70-kDa protein, which precedes the expression of 2',5'-oligoadenylate synthetase; moreover, the transient nature of the synthesis of the hsp 70 in these cells is similar after both heat shock and ethanol treatment.

2',5'-Oligoadenylate synthetase expression is induced in response to heat shock.

Hyperthermia (45 degrees C) induced the synthesis of a characteristic heat-shock protein of 70,000 daltons (70 hsp) in Madin-Darby bovine kidney (MDBK) cells. In addition, subsequent to heat shock, there was a substantial increase in the 2',5'-oligoadenylate synthetase (2',5'-A synthetase) activity in both MDBK and human WISH cells. However, in contrast to 70 hsp synthesis, which reached its maximum 3 h after cell transfer from 45 to 37 degrees C, increase in 2',5'-A synthetase expression, conspicuous after 6 h, attained its maximum only 18 h after transfer. Another interesting observation is that, during recovery at 37 degrees C, the cells released into the medium heat-shock-induced factor(s) (HSIF) capable of inducing an increase in 2',5'-A synthetase activity in fresh MDBK cells. HSIF behaves as a polypeptide with a molecular weight of more than 5,000; it is relatively heat stable and sensitive to acidic treatment. HISF seems different from interferon (IFN) since: 1) no detectable antiviral state developed after infection in cells treated with HSIF; 2) antibovine IFN antibodies did not abolish the inducing capacity of HSIF; 3) IFN had an additive effect on the inducing capacity of HSIF, and 4) HSIF released from bovine cells induced a net enhancement of 2',5'-A synthetase activity in human WISH cells. The first three of these observations applied also to heat-shocked MDBK cells.

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

[Induction of atypical interferon after heat shock]. / Induction d'un interféron atypique après choc thermique.

Hyperthermia (45 degrees C) induced the release of heat-shock induced factor(s) (HSIF) in the culture medium of Madin-Darby bovine kidney cells (MDBK) during their recovery period at 37 degrees C. HSIF is capable of inducing an increase in the 2'5' oligoadenylate synthetase activity in fresh MDBK cells without any detectable antiviral activity (Chousterman et al. J. Biol. Chem. 1987, 262, 4806-4811). We demonstrated here that even though HSIF did not crossreact antigenically with alpha, beta or gamma bovine interferons, it was still also able to induce an antiviral activity which copurified with its capacity of inducing 2'5' oligoadenylate synthetase activity. Therefore we concluded that HSIF is an atypical bovine interferon induced in response to heat shock.

Paracrine activities of porcine trophoblastic interferons.

Interferon-gamma (IFN-gamma) and a type I IFN (spI IFN) are transiently coexpressed by trophoblastic cells of pig conceptuses at implantation between day 12 and day 20 of gestation. The local effects of these trophoblastic IFNs were examined on endometrial cells and on trophoblast by measuring antiviral activity and the induction of (2',5')-oligoadenylate synthetase activity. Trophoblastic vesicles were shown to be susceptible to infection by vesicular stomatitis virus and transmissible gastroenteritis virus. Vesicular stomatitis virus multiplied by about 1000 times in trophoblastic vesicles, and endogenous trophoblastic IFNs or exogenous recombinant IFN-gamma or spI IFN had no effect on virus production. No (2',5')-oligoadenylate synthetase activity could be measured on the trophoblast, even after treatment with IFN-gamma or spI IFN. These results clearly show that trophoblastic IFNs cannot induce antiviral resistance or (2',5')-oligoadenylate synthetase activity in the trophoblast, suggesting that these IFNs have no autocrine function. Endometrial epithelial and stromal cells in primary cultures displayed distinct sensitivity to the antiviral effect of IFN-gamma and spI IFN. Stromal fibroblasts were highly sensitive to spI IFN but weakly sensitive to IFN-gamma; epithelial cells were sensitive to both IFNs. The same sensitivity pattern was obtained when measuring the (2',5')-oligoadenylate synthetase activity. Flushing fluid, containing IFN-gamma and type I IFN, was a potent inducer of antiviral effect and (2',5')-oligoadenylate synthetase activity. It is therefore postulated that the endometrial epithelium is the most likely target of trophoblastic IFNs. It is possible that these IFNs play a role in the viral protection of conceptuses.

Interferon inducibility and sensitivity of human teratocarcinoma-derived cell lines.

Three cell lines tera I, tera II, and PA1, derived from human teratocarcinomas were tested for their capacity to produce interferon (IFN) and for their sensitivity to both human IFN-alpha and IFN-beta. When treated with Newcastle disease virus or Sendai virus, or a synthetic polyribonucleotide, poly(rI):poly(rC), tera I cells produced no IFN and the 2',5'-oligoadenylate (2-5A) synthetase enzymatic pathway was not activated, although there was an increase in protein kinase. In contrast, tera II and PA1 cells produced IFN and both enzymatic activities were detected. IFN treatment has no effect on the growth of any of the cell lines. Tera I and PA1 cells did not develop resistance to challenge with vesicular stomatitis virus or encephalomyocarditis virus, but the growth of a type-C baboon retrovirus was inhibited. Tera II cells were protected against all three viruses. It appears that human teratocarcinoma cell lines can thus differ greatly in their ability to produce IFN and to respond to it.

Lack of in vivo activation of the interferon system in HBsAg-positive chronic active hepatitis.

The in vivo activation state of the interferon system was biochemically evaluated in patients with HBsAg-positive liver disease by assaying the interferon-induced enzyme, 2'5'-oligoadenylate synthetase, in peripheral blood mononuclear cells. All patients with chronic active hepatitis had normal levels of enzyme activity. Increased values were found in 77% of patients with acute hepatitis, 50% of those with chronic persistent hepatitis and 54% chronic healthy carriers. These results provide evidence for lack of activation of the interferon system in HBsAg-positive chronic active hepatitis and support the hypothesis that an in vivo defective interferon response may aid in development of chronic active hepatitis.

The interferon compartment of the immune response in human malaria: II. Presence of serum-interferon gamma following the acute attack.

The present study concerns the monitoring of serum-interferon (serum-IFN) levels among 189 patients followed after and sometimes during an acute episode of malaria due mainly to Plasmodium falciparum (P. falciparum). Of these patients, 110 known to have no other parasitic or infectious disease were followed in France; 79 were from Thailand, among which 25 cases of neuromalaria were diagnosed. In a first four-month survey conducted in France, among 100 patients seen after the acute attack, serum-IFN-gamma was characterized among 87% cases for which at least two sera were controlled, whereas in a healthy population no serum-IFN was present. When efforts were concentrated on screening ten cases during the first 48 h of the febrile attack, serum-IFN-alpha was mainly characterized, whereas serum-IFN-gamma was present only once. Elevated leukocyte 2',5' oligoadenylate synthetase levels were found among several IFN-alpha positive patients of this study group. A peculiarity pertaining to the patients from Thailand was that one-third (25 cases) were cerebral malaria cases. Among these, 15 were followed under hospitalization during the first 96 h. In this study group, the onset of circulating immune interferon was found to be preceded or accompanied by that of IFN-alpha. Thus, if serum-IFN-gamma is largely characterized among malaria patients followed after the acute attack, it is possible that the onset of circulating immune interferon is generally preceded by that of IFN-alpha.

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).

Tenoxicam, a non-steroid anti-inflammatory drug, is unable to increase the response rate in patients with chronic hepatitis C treated by alpha interferon.

The purpose of this study is to compare a combination of interferon (IFN)-alpha2a (Roferon) + Tenoxicam with IFN-alpha2a alone in the treatment of chronic hepatitis C. This prospective, randomized double-blind study included 149 patients, all of whom were diagnosed with active chronic hepatitis C but non-cirrhotic (ALT > or = 1.5 upper limit of normal, anti-hepatitis C virus (HCV) positive by enzyme-linked immunosorbant assay2 and RIBA3). The patients were randomized in two groups, as follows: G1 (n = 76): IFNalpha2a 3 million units times per week during 6 months + placebo; and G2 (n = 73): IFNalpha2a 3 million units three times per week + Tenoxicam (20 mg/day) during 6 months. Alanine aminotransferase (ALT) and HCV RNA were determined before and at months 6 and 12 of treatment. 2'5' oligoadenylate synthetase activity (2'5' AS) was dosed in mononuclear cells before and at 3-month treatment intervals in 28 patients. Liver biopsy was performed before and 6 months after the end of therapy. Parameters were similar before therapy for both groups. Biochemical and virological responses were similar for both groups at month 6 (49.3% vs. 42.9% and 43.3% vs. 38.3%, respectively) and month 12 (28.3% vs. 23.8% and 17.2% vs. 17.5%, respectively). HCV RNA level significantly decreased in both groups at month 6, with no difference whatever the therapy; however, the HCV RNA level returned to initial values at month 12 and was the only significant prognostic factor of a sustained response. No peak of 2'5' AS activity was observed during treatment in patients with dual therapy. A histological improvement was also noted in both groups without difference, regardless of therapy. The percentage of adverse events was identical for both groups. Paracetamol intake, assessed in 80 patients, was 49.1 g per 6 months in the G1 group and 22.5 g per 6 months in the G2 group (not significant). In conclusion, the non-steroid anti-inflammatory drug, Tenoxicam, does not increase IFNalpha efficacy in the treatment of chronic hepatitis C. This combination is well tolerated and partially lowers Paracetamol intake, but not preexisting alpha-IFN adverse events.