High expression of exogenous cDNAs directed by HIV-1 long terminal repeat in human cells constitutively producing HIV-1 tat and adenovirus E1A/E1B.

A eukaryotic vector-host cell system is described where the additive transactivating effects of HIV-1 tat and adenovirus E1A on HIV-1 long terminal repeat (LTR) are exploited to increase expression of exogenous cDNAs. Human 143B and 293 cells, the latter constitutively producing E1A, were used as host cell lines. The bacterial gene chloramphenicol acetyltransferase (CAT) and the hepatitis B surface antigen (HBs-Ag) gene were employed as reporter genes inserted in pRPneoU3R, an episomal vector containing BK virus replication origin and early region, where cDNAs are expressed under control of HIV-1 LTR. The 293 cells were transformed by tat expression vectors to constitutively express tat. Stable cell clones of 293tat cells, constitutively expressing CAT after transformation with pRPneoU3R-CAT, show a CAT activity 600-fold higher than normal 293 transformed cells. CAT expression obtained in normal 293 cells can be transiently increased 10-fold by transfection by vectors expressing tat. The 293tat cells transformed by pRPneoU3R-HBs, an episomal vector expressing HBs-Ag from HIV LTR, yielded stable cell clones secreting HBs-Ag in the culture medium at a concentration up to 744 ng/ml or 44 ng/10(6) cells/24 h, 48-fold more than normal 293 cells. The use of this system for constitutive or inducible expression of sequences under control of HIV-1 LTR is discussed in view of possible applications for diagnostic, vaccinal and therapeutic purposes.

Development and evaluation of an ELISA using secreted recombinant glycoprotein B for determination of IgG antibody to herpes simplex virus.

An ELISA for the determination of IgG antibody to herpes simplex virus (HSV) was developed using a secreted recombinant HSV-1 glycoprotein B (gB-1s) as a solid phase. The clinical validity of the ELISA was established by testing different groups of sera containing HSV-1, HSV-2, or mixed antibody, in parallel with gB-1s ELISA and conventional HSV-1/HSV-2 ELISA. The new gB-1s ELISA detected HSV-1/HSV-2 antibody in sera from 48 subjects with either HSV-1 or HSV-2 past infection as well as in sera from 20 patients with primary infections by either serotype, in complete agreement with the results obtained using conventional ELISA. In 7 patients with HSV-1 encephalitis the kinetics of the gB-1s serum/cerebrospinal fluid antibody-titre ratio paralleled that of conventional ELISA over a period of time of up to 4 years. Acute and convalescent-phase sera from 28 patients with acute infections by human herpesviruses other than HSV did not show a significant cross-reactivity with gB-1s. In conclusion, gB-1s ELISA is a reliable assay for determination of HSV immune status as well as for diagnosis of both primary HSV-1 and HSV-2 infections and for diagnosis of HSV-1 encephalitis.

Carotenoid and ultrastructure variations in plastids of Arum italicum Miller fruit during maturation and ripening.

The changes in the pigment pattern and composition occurring in the Arum italicum berry during the various steps of maturation (ivory to deep-green stages) and ripening (yellow and red-orange stages) were studied and correlated to the ultrastructural modifications of plastids. Transmission electron microscopy showed that each stage was characterized by a specific plastidial type following the unusual sequence amyloplast-->chloroplast-->chromoplast. Plastidial transitions were accompanied by profound modifications in the pigmental composition, in particular, in the pattern of carotenoids and their precursors. The HPLC analysis of the carotenoids showed that, besides the two usual all-trans metabolic pathways leading to lutein through alpha-carotene and to auroxanthin through beta-carotene, an additional cis-isomeric biosynthetic pathway leading to cis-neoxanthin through cis-beta-carotene exists. During the pre-ripening stages, the three pathways were present even if with qualitative and quantitative variations. When complete ripening was reached, a block occurred at the cyclization level causing the accumulation of both all-trans (i.e. gamma-carotene and neurosporene) and cis-isomer (i.e. lycopene and zeta-carotene) carotene precursors. Because of the occurrence of unusual pigments and the presence of the three main plastidial types, the fruit of A. italicum may constitute a most instructive model for the study of carotenogenesis.

Constitutive expression in human cells of herpes simplex virus type 1 glycoprotein B gene cloned in an episomal eukaryotic vector.

Expression of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) was obtained in human cells from the gB-1 gene cloned in the episomal replicating vector pBK-1, which contains the origin of replication and early region of the human papovavirus BK. Selective systems for the TK+ phenotype in TK-143B cells and for resistance to G418 in adenovirus 5-transformed 293 cells were used to obtain stable transformants that produced gB-1. While gB-1 expression in 143B cells required induction by HSV-1 early proteins, constitutive gB1 production was observed in 293 cells, where endogenous trans-acting factors probably replace the need for early viral products in the activation of the cloned gB-1 gene. The amount of recombinant gB-1 was comparable to that produced during HSV-1 lytic infection in human cells, due to amplification of the inserted gene in the replicating episomal vector. Expression of gB-1 was induced by cadmium and zinc when the promoter of the mouse metallothionein-I gene was placed upstream of gB1 structural sequences. The inducible system where the gB-1 gene is under the control of its own promoter could be employed to clarify the role of early viral products in induction of gB-1 synthesis. Constitutive expression of gB-1 in human cells could provide useful material for diagnostic purposes and for the preparation of a subunit vaccine against HSV infections.

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.

Herpes simplex virus (HSV) glycoprotein H is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or ts mutants in the known HSV glycoproteins.

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gI, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein(s) is a property of gH.