Defective Friend Spleen Focus-Forming Virus: Interfering Properties and Isolation Free from Standard Leukemia-Inducing Helper Virus

Defective Friend spleen focus-forming virus (SFFV) is able to interfere with the ability of its naturally occurring leukemia-inducing helper virus (LLV-F) to induce XC plaque formation in several different strains of mouse embryo cells. This interference has been observed by using two different SFFV preparations, one contained in an NB-tropic stock of Friend virus (FV) complex, and the second present in a C57BL-adapted strain of FV complex containing an associated B-tropic LLV-F helper. The LLV-F in NB-tropic FV complex effectively induced XC plaques in C57BL/6 (Fv-1^bb; Fv-2^rr) mouse embryo fibroblasts (MEF) only in the absence of coinfecting SFFV, indicating that Fv-2-associated resistance to SFFV-induced focus formation in vivo does not necessarily extend to the restriction of SFFV function(s) in vitro (i.e., in Fv-2^rr C57BL MEF). SFFV interference appears to be an intracellular event since LLV-F can adsorb onto, penetrate, and rescue defective murine sarcoma virus (MSV) from transformed 3T3FL S⁺L⁻ cells with equal efficiency in the presence and absence of SFFV. However, significantly fewer LLV-infected S⁺L⁻ cells released LLV-F progeny if SFFV was present. These observations suggest that Friend SFFV may be classified as a defective, interfering (DI) particle. Further support for this conclusion has come from studies designed to investigate two physical properties of defective SFFV particles. SFFV layered onto a 0 to 20% sucrose sedimentation gradient was recovered as a symmetrical band of virus that sedimented more slowly than standard LLV-F particles. Pooled SFFV-containing gradient samples contained visualizable type C virus particles and occasionally small amounts of detectable LLV-F. In an attempt to determine the buoyant density of sedimentation gradient-purified SFFV, pooled SFFV samples were layered onto a 25 to 50% sucrose equilibrium density gradient and were centrifuged to equilibrium. Greater than 50% of the infectious SFFV originally layered onto this gradient was recovered and seen as a narrow symmetrical band with peak SFFV infectivity at a sucrose density of 1.14 g/ml. The observed difference between SFFV and LLV-F buoyant densities appears to be related to an inherent physical property of each virus. Mixtures of these two viruses express the buoyant density of that virus population which is in excess in fabricated FV complexes probably due to the formation of SFFV-LLV aggregates. Finally, gradient-purified SFFV failed to induce XC plaques in MEF and did not function to rescue MSV as expected since SFFV itself is replication defective.     

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice (for a review, see reference 42). Friend SFFV is a replication-defective virus with deletions in its env gene, giving rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity to the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice (49). The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (18, 37), as follows: the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases (11-12). Susceptibility to SFFV-induced disease is associated with expression of a short form of the receptor tyrosine kinase Stk, termed sf-Stk, that is transcribed from an internal promoter within the Stk gene of Fv-2-susceptible (Fv-2^ss) mice but not Fv-2-resistant (Fv-2^rr) mice (37) and is abundantly expressed in erythroid cells (11). Infection of erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemia-inducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation (42). Previous studies demonstrated that this Epo-independent erythroblastosis is due to the cell surface interaction of the SFFV envelope protein with the Epo receptor (EpoR) and sf-Stk (31). While interaction with the EpoR appears to be responsible mainly for the induction of Epo-independent differentiation (52), Epo-independent erythroid cell proliferation depends upon activation of sf-Stk. We recently demonstrated that sf-Stk covalently interacts with SFFV-P gp55 in hematopoietic cells that express the EpoR and that this interaction induces sf-Stk activation (31). Furthermore, exogenous expression of sf-Stk, but not a kinase-inactive mutant of sf-Stk, in bone marrow cells from sf-Stk null mice can restore Epo-independent erythroid colony formation in response to SFFV infection (5, 41). Thus, the SFFV envelope glycoprotein induces Epo-independent proliferation of erythroid cells mainly by activating sf-Stk. While sf-Stk is a key susceptibility factor for erythroblastosis induced by both SFFV-P and SFFV-A (18), it is not required for the induction of erythroblastosis by the SFFV mutant BB6, which encodes an envelope glycoprotein, gp42, that is deleted in the membrane-proximal extracellular domain (19) and does not induce sf-Stk activation (31). gp42 of SFFV-BB6 appears to exert its biological effects on erythroid cells by efficiently interacting with the EpoR (9). Compared with wild-type SFFV, SFFV-BB6 causes a relatively indolent and slowly developing disease in mice (19).A number of signaling pathways normally activated in erythroid cells after erythropoietin (Epo) binds to its cell surface receptor (40) are constitutively activated in erythroid cells infected with SFFV. These include JAK/STAT, Ras/Raf/mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, and the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathways (24, 25, 28-30, 32). SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17, 31, 43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8, 20, 21) or by SFFV through sf-Stk (5, 14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5, 29). The class IA subclass of PI3-kinase is a heterodimer comprising the p110 (α, β, δ) catalytic unit and one of five regulatory subunits (85α, p55α, p50α, 85β, and 55γ) (15). The first 3 regulatory subunits are all splice variants of the same gene (pik3r1). Deletion of pik3r1, which encodes p85α, p55α, and p50α, is lethal (6, 7), and these regulatory subunits of PI3-kinase are required for normal murine fetal erythropoiesis in mice (10).To determine the role of p85α in SFFV-induced erythroleukemia, we used a distinct nonlethal pik3r1 knockout mouse which lacks only the p85α regulatory subunit of PI3-kinase (45, 47), allowing the study of SFFV-induced erythroleukemia in adult mice. Our results indicate that p85α regulates SFFV-induced erythroid hyperplasia induced in vivo by sf-Stk-dependent, but not sf-Stk-independent, isolates of the virus as well as stress-induced erythropoiesis and suggest that this regulation may occur through the interaction of sf-Stk with p85α.     

Immunological Properties of Friend Virus from Mouse Spleen Obtained by Gel Filtration

Gel filtration, with Sephadex G-200, was utilized to obtain Friend virus (FV) from infected spleen homogenate. Highly infectious and immunologically active material was eluted as a large initial protein peak, whereas hemoglobin and other noninfectious materials were found in two subsequent peaks. Fractions that made up the first peak were reactive in serological tests with antiserum towards FV. Formalinized fractions from the first peak, when combined with Freund's complete adjuvant, were able to protect mice against subsequent FV challenge. Further purification of antigen was carried out by sucrose gradient ultracentrifugation.     

Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

Sequence Flexibility in the Polytropic env gp70-Derived Region of the Membrane Glycoprotein (gp55) of Friend Spleen Focus-Forming Virus Affects Its Biological Activity

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132–137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV_MS), which encodes a mutant gp55 membrane glycoprotein with an ecotropic env gp70 sequence, was nonpathogenic. Here we injected the F-SFFV_MS–Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic env-specific probe. Cloning and DNA sequence analysis of the env region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV_MS env gene by a homologous recombination with the F-MuLV clone 57 env gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic env genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.Friend spleen focus-forming virus (F-SFFV), a replication-defective mouse type C retrovirus contained in the Friend virus complex, causes an acute erythroleukemia in adult mice of susceptible strains in the presence of a helper virus, such as the replication-competent Friend murine leukemia virus (F-MuLV) (reviewed in references 8 and 23). F-SFFV does not encode a viral oncogene, but its defective env gene product, gp55, plays a critical role in inducing the disease. In vitro biochemical evidence demonstrated that gp55, a membrane glycoprotein encoded by the polycythemia-inducing isolate of F-SFFV (F-SFFVp), specifically binds to a mouse erythropoietin receptor (EPO-R) and activates it, causing mitogenic signal transduction in the absence of the natural ligand erythropoietin (EPO) (12). Since the EPO-R is expressed in the erythroid progenitor cells, and the interaction between EPO and EPO-R regulates the level of erythropoiesis (15), it is assumed that the continuous expression of F-SFFV gp55 results in an abnormal proliferation of these cells, leading to leukemic transformation after several additional cytogenetic changes (3).gp55, although closely related to the Env protein of MuLV, is not incorporated into the retrovirus particles, but stays inside the cells, mainly in the rough endoplasmic reticulum membrane, with a small fraction of the molecules (3 to 5%) processed through the Golgi apparatus to the cytoplasmic membrane (6, 21, 24). Cell surface-localized gp55 is then shed from the cells, probably after proteolytic cleavage from the transmembrane domain (6, 20, 21).There are three major differences between the primary structures of gp55 of F-SFFVp and the Env protein of ecotropic MuLV (1, 4, 31). These are, from the N terminus, a substitution by the polytropic (dualtropic) env gp70 sequence, a 585-bp deletion which eliminates the proteolytic cleavage site for gp70 and p15E, and a 6-bp duplication and a single base insertion which cause premature termination of translation and loss of a 34-amino-acid peptide at the C terminus. By constructing F-SFFV encoding a mutant gp55, analyzing its pathogenicity in vivo, and also obtaining spontaneous revertant F-SFFVs from the mutant F-SFFV, we demonstrated that each of these three structural differences is essential for the pathogenicity of gp55 (2, 27–29). We previously reported (28) that the constructed F-SFFV (F-SFFV_MS) encoding the mutant gp55, in which the polytropic gp70 sequence had been replaced by the ecotropic F-MuLV clone K-1 gp70 sequence, was nonpathogenic in adult mice. The polytropic gp70-derived sequence in gp55 appears to contain a binding site for EPO-R (32). The purpose of the present study was to obtain spontaneous revertants from mice neonatally injected with F-SFFV_MS and to analyze the structure of their gp55s, with the goal of pinpointing the sequence required for pathogenicity and activation of EPO-R. The results indicated the importance of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

Studies with aphidicolin on the Fv-1 host restriction of Friend murine leukemia virus.

The murine gene Fv-1 exerts a major control over the replication of Friend murine leukemia virus (F-MuLV). An effect of the gene product has been determined to be at the level of accumulation and integration of viral DNA. Aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, was studied in murine cells infected either permissively or nonpermissively with regard to the Fv-1 genotype. Results indicated that inhibition of DNA polymerase alpha did not affect the accumulation of form III viral DNA in either permissive or nonpermissive cells. However, the normal accumulation of circular form I DNA in permissive cells was inhibited. The block in the accumulation of form I DNA resembled that occurring in some F-MuLV Fv-1-nonpermissive infections. Additionally, aphidicolin treatment resulted in the accumulation of novel low-molecular-weight viral DNA species, normally detectable in a nonpermissive infection of NIH cells with B-tropic F-MuLV. These data suggest that the Fv-1 gene product may interact with host DNA polymerase alpha to prevent viral replication.     

Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

The murine gene Fv-1 predominantly controls the outcome of infection by murine ecotropic retroviruses. The inhibition of virus replication by the Fv-1 gene product has been determined to be at an early stage in virus replication. Mechanistically, its effect appears to be on the accumulation of unintegrated proviral DNA or its integration or both. We investigated the synthesis of unintegrated proviral DNA, using several clones of B-, N-, or NB-tropic Friend murine leukemia virus. Our results indicate that the accumulation of B-tropic proviral DNA in NIH cells may be inhibited at either the level of linear (form III) or covalently closed circular DNA (form I), depending upon the degree of restriction of the clone of virus used. We confirmed that there is an effect of the Fv-1 gene on the accumulation of form I DNA of either B- or N-tropic Friend murine leukemia virus. However, the decrease in infectious centers effected by the Fv-1 gene did not correlate quantitatively with the effect on form I proviral DNA produced by N-tropic Friend murine leukemia virus in nonpermissive cells. Lastly, we demonstrated in nonpermissively infected NIH cells that a rapidly migrating doublet of viral DNA is formed.     

Effect of BCG on Friend Disease Virus in Mice

NONSPECIFIC stimulation of the reticulo-endothelial system and resistance against tumorigenesis are produced by injection of attenuated Mycobacterium bovis (BCG). Biozzi et al.¹ demonstrated resistance to Ehrlich's ascites tumour in BCG-immune mice and Halpern et al.² showed that such immunization effected control of T-8 tumours in rats. Further, at least partial control of other tumours^3–6 has been effected by this procedure in mice and hamsters.     

Effect of the Fv-1 locus on the titration of murine leukemia viruses.

Titration of N- and B-tropic murine leukemia viruses on sensitive and resistant cell lines has been studied by direct XC plaque assay and infective center assay. The titration of cloned B-tropic virus by infective center assay on BALB/3T3 (Fv-1b/b) and NIH/3T3 (Fv-1n/n) cells gave one-hit patterns, with 100-fold less infected NIH/3T3 cells than BALB/3T3 cells. The titration of B-tropic virus on DBA/2 cells (Fv-1n/n) was also a one-hit. The titration of a one-hit curve, and there were about 100-fold less infected BALB/3T3 cells than NIH/3T3 cells. Comparable results were obtained by titrating the cloned N-tropic virus on congenic SIM (Fv-1n/n) and SIM.R (Fv-1b/b) cells or the Gross N-tropic virus on BALB/3T3 cells. Therefore, our data indicate that the multiple-hit phenomenon described previously may not be an essential part of the Fv-1 gene restriction.     

Development of a Gene Transfer System for the Mycelia of Flammulina velutipes Fv-1 Strain

To develop a gene transformation method for Flammulina velutipes, we constructed a vector with hph gene under control of the trp1 gene promoter. The vector was integrated into protoplast derived from mycelia by the calcium-polyethylene glycol method, as it has not been reported for F. velutipes. Transformation efficiency was much improved when transformation was performed by the restriction enzyme mediated integration method.     

LCB1基因表达对酵母神经酰胺合成的影响

酿酒酵母(Saccharomyces cerevisiae)LCB1（Long chain base）基因被克隆到酵母诱导表达载体pYES2中,并转入到FY2中,用半乳糖诱导表达。与对照相比,质粒所含LCB1基因的表达,使酵母细胞干重略有下降,而神经酰胺的含量提高为对照的1.9倍。     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Effect of Chemicals on the Early Stages of Tobacco Mosaic Virus Protein Polymerization

Osmotic pressure and sedimentation velocity techniques have been used to investigate the effect of KSCN, thiourea, EDTA, acetamide, and sucrose on the 53,000 molecular weight A protein at pH 6.5-7.0. In the presence of all the compounds except thiocyanate, the number average molecular weight lies between 50,000 and 56,000 which corresponds to a trimer of three chemical subunits. In the presence of thiocyanate, the molecular weight decreases initially sharply with increasing concentration of thiocyanate to 0.1 M, then dissociation proceeds with less efficiency with increasing concentration of KSCN until a molecular weight close to a monomer (21,700) is obtained at 0.59 M KSCN. Sedimentation data agree with osmotic pressure data since, in the absence of thiocyanate, the measured values of S_20,w indicate that the favored state is that of a trimer. In the presence of thiocyanate, however, S_20,w decreases with thiocyanate concentration to S_20,w = 1.9, which is the value reported for the monomer.     

鳜鱼传染性脾肾坏死病毒的胞嘧啶5-甲基转移酶基因结构及其序列分析

对鳜鱼传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）的胞嘧啶5－甲基转移酶（MTase）基因的结构及序列进行了分析。序列比较分析表明,ISKNV MTase编码区全长684bp,编码长227个氨基酸的蛋白质,推测分子量为25855D。与一些细菌的MTase比较,ISKNV MTase也含有负责转移甲基的4个保守区,但缺乏识别靶序列的保守区。比较ISKNV与其它6种脊椎动物虹彩病毒的MTase序列并建立系统树,ISKNV显著不同于蛙病毒属和淋巴囊肿病毒属。7种脊椎动物虹彩病毒MTase具有高度保守区,可以此设计引物用PCR方法鉴定脊椎动物虹彩病毒。     

Ribavirin: Biotechnological Synthesis and Effect on the Reproduction of Vaccinia Virus

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied on the culture of Vero cells. The combination of ribavirin and vidarabin was shown to provide the antiviral effect at lesser concentrations than with these compounds taken separately.