Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Studies of the Epstein-Barr virus receptor found on Raji cells. I. Extraction of receptor and preparation of anti-receptor antibody

Raji, a human lymphoblastoid cell line, expresses a membrane receptor (EBVR) specific for Epstein Barr virus (EBV). A component that binds EBV was extracted from this cell line by treatment of the cells for 3 hr on ice with Tris buffer containing 10% glycerol. The treatment reduced the capacity of the cells to bind virus, and after concentration the receptor extract (RE) inhibited both EBV binding and superinfection of fresh Raji cells. Similarly prepared extracts of EBVR- cells lacked such activity. An antibody was made to the extract (anti-RE), which after absorption with EBVR- cells, bound to the same percentages of EBVR+ lymphoblastoid cell lines, EBVR+ human/mouse somatic cell hybrids, and fresh peripheral B cells as the virus did. In reciprocal assays, preincubation of EBVR+ cells with anti-RE inhibited virus binding. Doubly stained patches were observed on membranes of EBVR+ cells that had been incubated simultaneously with virus and anti-RE and stained respectively with rhodaminated and fluoresceinated reagents. The major polypeptide immunoprecipitated by anti-RE from radiolabeled Raji cells had an approximate calculated m.w. of 150,000.     

Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.     

Delayed cytosolic exposure of Japanese encephalitis virus double-stranded RNA impedes interferon activation and enhances viral dissemination in porcine cells

Interferon is a principal component of the host antiviral defense system. In this study, abortive focus formation by Japanese encephalitis virus (JEV) in primate cells was accompanied by early interferon induction, while productive focus formation in porcine cells was associated with a late interferon response. Neutralization antibodies against interferon relieved the restricted infection in primate cells, and increasingly larger foci were generated as treatment with exogenous interferon was delayed, thereby establishing a solid correlation between interferon response and viral dissemination. However, delayed interferon induction in JEV-infected porcine cells occurred in the absence of active inhibition by the virus. We further demonstrated that JEV mediates interferon activation through double-stranded RNA and cytosolic pattern recognition receptors. Immunofluorescence and subcellular fractionation studies revealed that double-stranded RNA is concealed in intracellular membranes at an early phase of infection but eventually appears in the cytosol at later periods, which could then allow detection by cytosolic pattern recognition receptors. Interestingly, cytosolic exposure of double-stranded RNA was delayed in porcine cells compared to primate cells, independent of total double-stranded RNA levels and in correlation with the timing of the interferon response. Furthermore, when double-stranded RNA was artificially introduced into the cytosol of porcine cells, more rapid and robust interferon activation was triggered than in viral infection. Thus, cytosolic exposure of JEV double-stranded RNA is imperative for interferon induction, but in cell lines (e.g., porcine cells) with delayed emergence of cytosolic double-stranded RNA, the interferon response is late and viral dissemination is consequently enhanced.     

The use of a complementary DNA probe to detect accumulation of mengo RNA in infected cells pretreated with interferon.

Complementary DNA (cDNA) from Mengo virus RNA has been synthesized and used as a probe to measure the synthesis and accumulation of viral RNA in Mengo infected L cell cultures, treated or untreated with interferon. Under experimental conditions used (200 units interferon/ml and 50 virus plaque-forming units/cell) results show that there is some synthesis of Mengo virus RNA in cells treated with interferon. One hour after infection, treated cells contain three times less viral RNA than untreated cells; five hours after infection, this difference has increased to ten fold. As in the control, no fragmented Mengo virus RNA molecules were found in interferon treated cells. The smaller recovery of infectious particles from interferon treated cells as compared to RNA accumulation suggests that not only RNA accumulation is inhibited but also a step posterior in viral maturation.     

Antiviral and anticellular effects of interferon on the mouse embryonic cells transformed by viruses and/or chemical carcinogen.

The antiviral and anticellular activity of partially purified mouse interferon has been tested in cell lines transformed with Simian Virus 40 (MEB-SV 40), mouse sarcoma virus (Harvey strain) (MEB-MSV), and 20-methylcholanthrene (MEB-MCH), respectively. The transformed lines were derived from C3H mouse embryonic primary cells. It has been shown that the MEB-MSV cells were 10 to 50 times less sensitive to the antiviral effect of interferon than the MEB-SV 40 or MEB-MCH cells. A 30% reduction of the number of treated cells as compared with untreated control cells was taken as basis for comparison of anticellular activity of interferon in transformed lines. While the MEB-MCH cells required 1000 units of interferon for a 30% growth inhibition, about 3000 units were necessary for a comparable suppression of MEB-SV 40 cells and/or MEB-MSV cells.     

Interferonogenic and antiviral activity of the double-stranded replicative form of phage f2 RNA in tissue culture and on animals]

Biological activity of a new natural interferon inductor, the replicative RNA form of phage f2 (RFf2) was studied. A possibility of using RFf2 for production of highly active interferon under conditions of superinduction providing an increase in the interferon yield by to 256--512 times as compared to the control samples was shown. The protective interferonogenic and antiviral effect of RFf2 in mice infected with Semliki forest virus (SFV) and tickborne encephalitis virus (TBEV) was studied on administration of the inductor by various routes. It was found that intraperitoneal administration of RFf2 in a dose of 10 gamma per a mouse protected the infected animals from death. It was accompanied by production of up to 1280 units/ml of interferon in the blood serum of the animals. Maximum protection of the animals from death under conditions of the experiment (80 per cent on infection with SFV and 65 per cent on infection with TBEV) was observed when the preparation was administered twice: 4 hours after the infection. Combined use of RFf2 with chemotherapeutics (rimantadine) increased the protective effect both in the tissue culture and in vivo.     

Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

Antiviral activity of preparations of RNA isolated from cells treated with interferon (messenger RNA for an antiviral protein?)]

Chick embryo cultures treated with interferon yielded a biologically active RNA which, upon inoculation into chick embryo cells, created an antiviral condition in them. The level of vesicular stomatitis virus reproduction in such cells was 2-30% of that observed in the cells treated with control RNA. The maximum activity of the experimental RNA was seen 3 hours after the treatment with interferon.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

Protein kinase PKR plays a stimulus- and virus-dependent role in apoptotic death and virus multiplication in human cells

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-alpha)-induced apoptosis was comparable between the HeLa lines. NF-kappaB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-alpha. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.     

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced my human leukocytes and by E. coli

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.     

Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity

Recombinant human leukocyte interferon synthesized by Escherichia coli possesses antiproliferative activity in addition to antiviral activity. When the ability to inhibit multiplication of lymphoblastoid Daudi cells was examined, the growth-inhibitory capacity of recombinant leukocyte interferon was equivalent to that exhibited by crude human leukocyte interferon or by the homogeneous gamma 2 species of leukocyte interferon synthesized by human cells.     

Mx gene control of interferon action: different kinetics of the antiviral state against influenza virus and vesicular stomatitis virus.

The allele Mx regulates the extent to which interferon alpha/beta inhibits the growth of influenza viruses in mouse cells such as peritoneal macrophages. The time course of induction of the antiviral state against an influenza A virus is comparable in macrophages with and without Mx and is similar to that found with vesicular stomatitis virus. In contrast, the decay of the antiviral state against influenza virus is markedly slower in Mx-positive cells and slower than that against vesicular stomatitis virus observed in either Mx-positive or Mx-negative cells. Thus, after removal of interferon alpha/beta, Mx-positive cells remain protected against influenza virus at times when they have lost protection against vesicular stomatitis virus. These results suggest that interferon alpha/beta treatment activates different antiviral mechanisms, each acting against distinct groups of viruses and each independently controlled by host genes.     

Oligo(2'-5')adenylate synthetase in human lymphoblastoid cells

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)_nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)_nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.