Orgad Laub

Using high titer West Nile intravenous immunoglobulin from selected Israeli donors for treatment of West Nile virus infection

BackgroundWest Nile Virus (WNV) is endemic in Israel and a significant level of antibodies is present in the population due to natural exposure. Anecdotal cases suggested that the presence of anti-WNV antibodies in intravenous immunoglobulin (IVIG) from Israeli donors (IVIG-IL) assisted the recovery of patients with severe WNV infection.MethodsTo enhance the therapeutic efficacy of IVIG-IL against WNV infection, OMRIX Biopharmaceuticals, Israel, have developed a strategy for selection of plasma units from a 10% fraction of Israeli blood donors with anti-WNV antibodies. Positive units were processed into pharmaceutical grade WNV IVIG (WNIG). Following inoculation with WNV, mice received i.p. injections of different doses (0.01–8 mg/mouse) of IVIG-IL or WNIG, according to the specific experimental protocol.ResultsWNIG was about 10 times more potent (per gr of IgG) than was regular IVIG-IL when tested by ELISA and neutralization assays. In a mouse lethal WNV infection model, prophylactic treatment with WNIG was at least 5–10-fold more potent as compared to treatment with IVIG-IL. Treatment with WNIG during active encephalitis, three or four days following WNV infection, had a significant protective effect. WNIG was also very effective in protecting immunosuppressed mice. Indeed, treatment of dexamethasone-immunosuppressed mice with 0.2 or 1.0 mg WNIG 4 h after virus infection, led to 100% survival.ConclusionIVIG produced from selected plasma donated in WNV endemic regions can be used to produce WNV IVIG with superior activity for therapeutic and prophylactic measures.

Corticosteroids stimulate hepatitis B virus DNA, mRNA and protein production in a stable expression system

The effect of corticosteroids (Dexamethasone) on hepatitis B virus was investigated in human hepatoblastoma cells stable transfected with recombinant HBV DNA. Dexamethasone was found to cause elevation of HBsAg, HBeAg and viral DNA production. HBV poly(A)+ RNA was significantly increased in cells treated with Dexamethasone. Furthermore, pulse labelled nuclear HBV RNA was also stimulated by Dexamethasone. These findings, suggest that corticosteroids enhance expression of viral gene products by stimulating HBV transcription.

The metabolism of SV40 RNA is associated with the cytoskeletal framework

Abstract We prepared the cytoskeletal framework of SV40-infected BSC-1 cells late after infection by extracting the cells with Triton X-100. The cytoskeletal framework obtained by this procedure carries more than 80% of the newly synthesized viral polyribosomes. Almost all the viral-specific cytoplasmic RNA is engaged in the formation of cytoskeletal-associated polyribosomes. The cytoskeletal framework harbors both the poly(A) + 19 S and 16 S late viral RNAs, as well as most of the poly(A) − viral RNA. The poly(A) − viral RNA sediments in sucrose gradients between 4 and 18 S representing heterogeneous species and comprises about 30–50% of the total virus-specific RNA in the cytoplasm. Based on pulse-chase experiments it is concluded that: (i) the poly(A) + viral RNA emerges from the nucleus and becomes associated with the cytoskeletal framework, (ii) the decay rate of the poly(A) + RNA species on the cytoskeletal framework is faster than that of the poly(A) − viral RNA, and (iii) both the poly(A) + and poly(A) − viral RNAs detach from the cytoskeletal framework and move to the soluble fraction.

The initiation of transcription of sv40 DNA at late time after infection

Abstract In vivo labeled RNA was purified from productively infected cells and in vitro labeled RNA was purified from transcriptional complexes of SV40. The purified RNAs were denatured and fractionated by sedimentation through sucrose gradients. Labeled RNAs of various lengths were hybridized with restriction fragments of SV40 DNA of a known order. In both cases the shortest RNAs hybridized with a fragment which spans between 0.67 and 0.76 map units and the hybridization with this fragment decreased with successively longer RNAs indicating that transcription initiates within this fragment or very close to it. Similar enrichment for this fragment was obtained using nascent RNA chains labeled in vitro with a short pulse. Electron microscopic analysis of transcriptional complexes of SV40 has revealed a substantial fraction with one short nascent RNA chain. The initiation site of the nascent chains was mapped at coordinate 0.67 ± 0.02. The accumulation of transcriptional complexes with short nascent chains, initiated at coordinate 0.67 ± 0.02, and the abundance of labeled nascent RNAs complementary to a fragment spanning between 0.67 and 0.76 map units could indicate the existence of an attenuator site in which RNA chain elongation is blocked, unless a stimulating factor is present which allows transcription to continue into a complete transcript.

Estrogen suppresses hepatitis B virus expression in male athymic mice transplanted with HBV transfected Hep G-2 cells

Hormones are known to regulate both viral and cellular genes. It has been shown previously that estrogen has an effect on liver gene transcription and mRNA stability. Sex hormones might have a role in the chronic persistence of hepatitis B virus (HBV) infection. In fact, there is a male preponderance in the incidence of chronic HBV infection, and HBsAg expression was reported to be much higher in male transgenic mice than in the females. We investigated the effect of estrogen on HBV gene expression and regulation in athymic mice bearing 2.2.15 cells, a human hepatoblastoma cell line derived from Hep G-2 transfected with HBV sequences. Both male and female mice were treated with estradiol after tumors could be observed. Episomal DNA was extracted from the tumors and hybridized with 32P-labelled HBV DNA. Southern blot and slot blot analyses demonstrated that male mice had higher expression of HBV DNA. Estrogen treatment suppressed HBV DNA expression in males, but had only a minor effect on females. HBeAg production in male mice was also inhibited by estrogen treatment. HBV RNA extracted from 2.2.15 cells showed 2-3-fold reduction following beta-estradiol treatment. Moreover, inhibition of HBV transcription by estrogen was demonstrated by an RNA pulse-labelling experiment. These data indicate that estrogen inhibits HBV expression in the in vivo model presented in this study. These results might contribute to a better understanding of the effect of sex hormones on the pathogenesis of HBV-induced liver disease.

Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response

The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.

Transcription of Simian virus 40 VI. SV40 DNA-RNA polymerase complex isolated from productively infected cells transcribed in vitro

SV40 DNA-RNA polymerase complexes were isolated from cells productively infected with SV40. These complexes sedimented in sucrose gradients at about 23–25 S. They displayed an active endogenous RNA synthesis. Nascent RNA chains elongated in vitro but no reinitiation was detected. Hybridization to RNA exhaustion was used to measure the complementarity of the in vitro elongated RNA to the early (E) and late (L) SV40 DNA strands. About 5% of the RNA was complementary to the E-strand and 95% was complementary to the L-strand. Similar hybridization with the E- and L-strands was also obtained with RNA extracted from cells 48 hr postinfection. The E- and L-strand transcriptions were inhibited to the same extent by low concentrations of α-amanitin, indicating that both are transcribed by RNA polymerase B. It is concluded that the in vivo strand selection control mechanism determining the frequencies of transcription from the E- and L-strand is continued during in vitro transcription.

In vitro regulation of human hepatitis B virus core gene transcription

In the present study we used a HeLa whole cell extract transcription system to map the transcription start sites and the minimal promoter of the hepatitis B virus core gene. Two initiation sites located at residues 1792 +/- 5 and 1817 +/- 5 were identified. The minimal upstream region essential and sufficient for transcription was defined to a 105-base pair DNA fragment. These results are identical to the in vivo mapping of the transcription start sites and the minimal core gene promoter. When in vitro transcription elongation was carried out in the presence of the anionic detergent Sarkosyl, known to enhance premature transcription termination (attenuation), two short transcripts (as well as two run-offs) were synthesized. Kinetic studies indicated that the short transcripts resulted from a block to transcription elongation and not from RNA processing. RNA mapping showed that the short attenuated transcripts indeed initiated at the two core gene initiation sites and both prematurely terminated at nucleotide 1966 +/- 5, defined as the attenuation site. This site is located in the attenuator RNA within a uridine-rich sequence preceded by a stable hairpin structure. Attenuation at the same site occurred when transcription of the core gene was directed by the Ad2 major late promoter (MLP) and when the poly(A) signal, which precedes the attenuation site, was mutated from TATAAA to TAGAAA. We suggest that the elongation block at nt 1966 +/- 5 in vivo exerts a dual function: first, it regulates the level of RNA by attenuation during the first cycle of transcription and, second, it acts as a termination site at the end of the primary RNA transcript.

Attenuation in the Control of Gene Expression in Animal Viruses

Viral structural proteins self-assemble to produce the capsid of the virion. For an efficient self-assembly process the structural proteins should be synthesized in optimal amounts. This could be accomplished by a mechanism which somehow couples transcription in the nucleus to translation in the cytoplasm. A mechanism of gene regulation which couples transcription and translation, termed “attenuation” exists in procaryotes. This control is manifested through the synthesis of a small “leader” peptide. Successful synthesis of this peptide leads to transcription termination, whereas in the absence of its synthesis, the RNA polymerase is allowed to continue transcription through the structural genes that follow the DNA sequence coding for the leader peptide. In the present communication we summarize the available information concerning transcription termination and attenuation in eucaryotes and we show that a mechanism resembling attenuation in procaryotes regulates the production of VP1, VP2 and VP3 in SV40 and gene expression in other animal viruses. In analogy to procaryotes the leader protein of SV40 late RNA (“agnoprotein”) stabilizes the RNA conformation, stem-and-loop structure followed by us, which leads to transcription-termination, while deficiency of the agnoprotein leads to stabilization of an alternative RNA conformation which allows the RNA polymerase to continue transcription through the structural genes. These observations show that RNA polymerase II responds to a transcription-termination signal similar to that to which the procaryotic polymerase responds and they are included in a model in which a feedback control mechanism regulates the transcription of the viral mRNAs in the nucleus and the translation of their encoded proteins in the cytoplasm. The model has striking similarities to the attenuation model in amino acid biosynthetic operons of bacteria suggesting that SV40 has exploited a procacryotic control mechanism and adjusted it to the eucaryotic environment.

Transcription of the cellular DNA sequences in a cloned host-substituted SV40 dna variant

Abstract The transcription of a cloned host-substituted SV40 genome of defined structure was studied in cells coinfected with wild-type virus and in in vitro reactions with Sarkosyl nuclear extracts (transcription complex preparations) of the coinfected cells. Evidence for the transcription of the monkey DNA sequences in substituted SV40 was obtained in both systems. Efforts to detect similar transcripts in uninfected cells, or in cells infected with wild type SV40 alone, were not successful. Both the highly reiterated and nonreiterated types of cellular DNA sequences (which are linked in the genome of the cloned substituted SV40 variant) were transcribed in the coinfected cells and the RNA transcripts were detected in the nuclear and in the cytoplasmic fractions. Relative to the amount of wild-type SV40 RNA, 40% of the RNA synthesized after in vitro incubation of transcription complex preparations, hybridized with substituted SV40 cellular DNA sequences. In contrast, only 15% of the nuclear RNA and 4% of the cytoplasmic RNA from intact cells hybridized with the cellular DNA derived from substituted SV40. The sucrose gradient sedimentation profile of the host-substituted SV40 RNA was uniquely different from and more heterogeneous than that of wild-type SV40 RNA. RNA homologous to the host DNA in the substituted SV40 variant was associated only with lighter (disomal and monosomal) ribosomal fractions.