Role of pseudorabies virus glycoprotein gI in virus release from infected cells.

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.     

The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.     

Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.

The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products.     

Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus.

The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 176 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.     

Expression and characterization of the thymidine kinase gene of African swine fever virus.

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.     

Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5′ and 3′ ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3′ NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.     

Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism.

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.     

Packaging signals in alphaviruses.

Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This implies the existence of an encapsidation or packaging signal which would be responsible for selectivity. Previously, we had identified a region of the Sindbis virus genome that interacts specifically with the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the 132-mer is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome. For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene. This is the same region that was proposed to contain the packaging signal for Semliki Forest virus genomic RNA.     

A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function.

Novel suppressor variants of conditionally lethal HSV-1 gamma34.5 deletion mutants have been isolated which exhibit restored ability to grow on neoplastic neuronal cells. Deletion of the viral gamma34.5 genes, whose products share functional similarity with the cellular GADD34 gene, renders the virus non-neurovirulent and imposes a block to viral replication in neuronal cells. Protein synthesis ceases at late times post-infection and the translation initiation factor eIF2alpha is phosphorylated by the cellular PKR kinase [Chou et al. (1990) Science, 252, 1262-1266; (1995) Proc. Natl Acad. Sci. USA, 92, 10516-10520]. The suppressor mutants have overcome the translational block imposed by PKR. Multiple, independent isolates all contain rearrangements within a 595 bp element in the HSV-1 genome where the unique short component joins the terminal repeats. This alteration, which affects the production of the viral mRNA and protein from the Us11 and Us12 genes, is both necessary and sufficient to confer the suppressor phenotype on gamma34.5 mutant viruses. HSV-1 thus encodes a specific element which inhibits ongoing protein synthesis in the absence of the viral GADD34-like function. Since this inhibition involves the accumulation of phosphorylated eIF2alpha, the element identified by the suppressor mutations may be a discrete PKR activator. Activation of the PKR kinase thus does not proceed through a general, cellular 'antiviral' sensing mechanism. Instead, the virus deliberately activates PKR and encodes a separate function which selectively prevents the phosphorylation of at least one PKR target, eIF2alpha. The nature of this potential activator element, and how analogous cellular elements could affect PKR pathways which affect growth arrest and differentiation are discussed.     

Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter.

On the basis of a previous finding that the 7.8-kb HindIII-O fragment of the human cytomegalovirus strain Towne genome is nonessential for viral replication, we constructed a vector, pKM, that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using this vector, we constructed Towne-strain-derived recombinant virus in which a chimeric lacZ gene fused to the simian virus 40 promoter and a poly(A) signal were inserted in place of the O fragment. Two types of recombinants were obtained which carried the chimeric gene in opposite directions, beta-Galactosidase (beta Gal) was produced throughout the infection cycle in human embryonic lung cells infected with these recombinants, and the rate of its synthesis in the early stages of infection was comparable to that of synthesis of a 65-kDa viral glycoprotein, one of the abundantly produced viral proteins. The chimeric lacZ gene introduced was stable and no lacZ- revertants have been observed so far.     

Regulation of eIF2alpha phosphorylation by different functions that act during discrete phases in the herpes simplex virus type 1 life cycle

Multiple herpes simplex virus type 1 functions control translation by regulating phosphorylation of the initiation factor eIF2 on its alpha subunit. Both of the two known regulators, the gamma(1)34.5 and Us11 gene products, are produced late in the viral life cycle, although the gamma(1)34.5 gene is expressed prior to the gamma(2) Us11 gene, as gamma(2) genes require viral DNA replication for their expression while gamma(1) genes do not. The gamma(1)34.5 protein, through a GADD34-related domain, binds a cellular phosphatase (PP1alpha), maintaining pools of active, unphosphorylated eIF2. Infection of a variety of cultured cells with a gamma(1)34.5 mutant virus results in the accumulation of phosphorylated eIF2alpha and the inhibition of translation prior to the completion of the viral lytic program. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. To determine if Us11 regulates late translation, we generated two Us11 null viruses. In cells infected with a Us11 mutant, elevated levels of activated PKR and phosphorylated eIF2alpha were detected, viral translation rates were reduced 6- to 7-fold, and viral replication was reduced 13-fold compared to replication in cells infected with either wild-type virus or a virus in which the Us11 mutation was repaired. This establishes that the Us11 protein is critical for proper late translation rates. Moreover, it demonstrates that the shutoff of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus, previously ascribed solely to the gamma(1)34.5 mutation, actually results from the combined loss of gamma(1)34.5 and Us11 functions, as the gamma(2) Us11 mRNA is not translated in cells infected with a gamma(1)34.5 mutant.     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.     

The Us9 Gene Product of Pseudorabies Virus, an Alphaherpesvirus, Is a Phosphorylated, Tail-Anchored Type II Membrane Protein

The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321–332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein.     

Association of the herpes simplex virus type 1 Us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides

The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components.     

非洲猪瘟病毒的非必需基因：真的可有可无吗？

非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）引起的一种出血性、致死性的猪烈性传染病。ASF在全球广泛传播,给养猪业造成重大的经济损失。ASFV基因组庞大,可编码150多种蛋白,一些非必需基因编码的蛋白与调控病毒毒力、复制和免疫逃逸等相关。通过删除ASFV毒力相关的非必需基因所构建的减毒株是当前比较有前景的疫苗,然而其安全性有待提高。系统地鉴定ASFV非必需基因及其功能,不仅有助于ASF基因缺失疫苗的研发,也有益于ASFV致病机制研究。本文对目前已鉴定的ASFV非必需基因及其功能研究进行了总结分析,着重讨论了影响ASFV毒力、调控病毒复制、参与免疫逃逸的非必需基因及其编码蛋白的功能,旨在加深对ASFV病原学的认识,为新的ASFV非必需基因的鉴定和功能研究提供参考。     

DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.