Construction, phenotypic analysis, and immunogenicity of a UL5/UL29 double deletion mutant of herpes simplex virus 2

A number of studies have shown that replication-defective mutant strains of herpes simplex virus (HSV) can induce protective immunity in animal systems against wild-type HSV challenge. However, all of those studies used viruses with single mutations. Because multiple, stable mutations provide optimal levels of safety for live vaccines, we felt that additional mutations needed to be engineered into a candidate vaccine strain for HSV-2 and genital herpes. We therefore isolated an HSV-2 strain with deletion mutations in two viral DNA replication protein genes, UL5 and UL29. The resulting double deletion mutant virus strain, dl5-29, fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells. Nevertheless, dl5-29 expresses nearly the same pattern of gene products as the wild-type virus or the single mutant viruses and induces antibody titers in mice that are equivalent to those induced by single deletion mutant viruses. Therefore, it is feasible to isolate a mutant HSV strain with two mutations in essential genes and with an increased level of safety but which is still highly immunogenic.     

New opportunities for development of safe, effective live virus vaccines

Effective vaccines are not available for most viral diseases. This situation may soon change when the full force of contemporary molecular biology is applied to immunoprophylaxis. In certain viral diseases, particularly those affecting the respiratory and gastrointestinal tracts, live attenuated vaccines are needed to confer effective protection. Until now the major obstacle to success has been genetic instability. It may be possible to construct stable, satisfactorily attenuated mutants by cloning viral DNA or RNA and then subjecting the cloned DNA to enzyme surgery to create viable deletion mutations. Modified cloned DNA derived from positive-strand viral RNA could then be transferred back into its virus by transfection of cells. Conversion of mutant cloned DNA into negative-strand RNA and transfer into its virus will require a more elaborate type of rescue.     

Nitric oxide as an endogenous mutagen for Sendai virus without antiviral activity

Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.     

Induction of mutations in mammalian cells by the action of live vaccinal strains of poliomyelitis virus

The attenuated strains' poliomyelitis virus induces mutagenesis in human and Chinese hamster cultured cells. The mutagenic action of the poliovirus is detected at chromosome and gene levels and directly depends on the multiplicity of cell infection. Possibilities for developing nonmutagenic antivirus vaccines are discussed.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

The Autographa californica M nucleopolyhedrovirus ac79 gene encodes an early gene product with structural similarities to UvrC and intron-encoded endonucleases that is required for efficient budded virus production

The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an ac79-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded virus (BV) production was reduced in the ac79-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The ac79-knockout virus-infected cells produced plaques smaller than those infected with control ac79-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in ac79-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the ac79-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the ac79-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of ac79-knockout virus-infected cells. Tubular structures were not observed for ac79 viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

Virus-associated human tumors: cervical carcinomas and papilloma viruses

The latest experimental data on the role of viruses in the origin of human tumors are discussed. This group of viruses consists of T-cell leukemia virus type 1 (HTLV 1), herpes viruses (HHV 8 and Epstein-Barr virus), hepatitis B virus, and human papilloma viruses. The most typical feature of this group of viruses is a very long latent period from the initial infection to the development of the disease that varies between 10 and 40 years. The mechanism of malignant cell conversion is specific for each viral type but is mainly associated with a disruption of functions of cellular genes participating in the control of cell division and proliferation. It can be a direct inactivation of tumor suppressor genes by their interaction with viral gene products (papilloma viruses), or a trans-activation of cellular genes modulating cell proliferation by viral gene products (hepatitis B virus and HTLV 1). Viruses play an initiative role and additional genetic changes in the genome of infected cells are necessary for complete expression of the oncogenic potential of the viral genes. Only these cells will give rise to a monoclonal cell population with uncontrolled proliferation. New approaches for the creation of vaccines against cancers associated with hepatitis B virus and papilloma viruses (hepatocellular carcinomas and cervical tumors, respectively) are in progress. These vaccines have been found to be effective in prevention of the disease in the experimental models and are now beginning to be used for human vaccination.     

Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant.

We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype.     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.     

Engineering large viral DNA genomes using the CRISPR‐Cas9 system

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus‐infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time‐consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat‐Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene‐ablated HSV but also gene knock‐in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein–Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.     

Mutagenic effects of gamma-rays on plasmid DNA in Escherichia coli

A purified and dried DNA of plasmid pKO482 (galK+) is 10 times more resistant to the inactivating action of 60Co-gamma-rays than that of lambda phage. gamma-Irradiation of the plasmid DNA induces forward mutations of galK, the frequency of which increases linearly with the dose. The efficiency of the mutagenic action of gamma-rays on the plasmid galK locus is 10(-12) per 1 rad and per 1 base pair. The mutagenic effect of gamma-radiation but slightly depends upon bacterial recA+ gene and upon the SOS-repair system induced by UV-irradiation of the recipient cells. It is assumed that the premutational lesions induced in the purified DNA by the direct effect of gamma-radiation are fixed into mutations by misreplication.     

利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction

Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.