Dominant-negative FADD rescues the in vivo fitness of a cytomegalovirus lacking an antiapoptotic viral gene

Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADD^DN) in an MCMV recombinant. FADD^DN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADD^DN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging

The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.     

Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.     

Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery

Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.     

Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(Luc)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(Luc) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.     

Visualization of the dynamics of gene expression in the living mouse

Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal.     

Isolation and genetic characterization of ethanol-resistant reovirus mutants.

To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame M35 is defective in growth in vivo

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.     

Genetic analysis of the attenuation phenotype of poliovirus type 1.

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.     

The Adenovirus L4-22K Protein Is Multifunctional and Is an Integral Component of Crucial Aspects of Infection

A variety of cellular and viral processes are coordinately regulated during adenovirus (Ad) infection to achieve optimal virus production. The Ad late gene product L4-22K has been associated with disparate activities during infection, including the regulation of late gene expression, viral DNA packaging, and infectious virus production. We generated and characterized two L4-22K mutant viruses to further explore L4-22K functions during viral infection. Our results show that L4-22K is indeed important for temporal control of viral gene expression not only because it activates late gene expression but also because it suppresses early gene expression. We also show that the L4-22K protein binds to viral packaging sequences in vivo and is essential to recruit two other packaging proteins, IVa2 and L1-52/55K, to this region. The elimination of L4-22K gave rise to the production of only empty virus capsids and not mature virions, which confirms that the L4-22K protein is required for Ad genome packaging. Finally, L4-22K contributes to adenovirus-induced cell death by regulating the expression of the adenovirus death protein. Thus, the adenovirus L4-22K protein is multifunctional and an integral component of crucial aspects of infection.     

Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.