Functional analysis of human cytomegalovirus UL/b' region using SCID-hu mouse model.

Human cytomegalovirus (HCMV) attenuated strains, Towne, and AD169, differ from prototypic pathogenic strains, such as Toledo, in that they are missing a ∼15-kb segment in the UL/b' region. In contrast to the attenuated strains, Toledo can replicate in human tissue implants in SCID (SCID-hu) mice. Thus, this model provides a unique in vivo system to study the mechanism of viral pathogenesis. Twenty-two ORFs have been annotated in the UL/b' region, including tissue-tropic genes encoded in a pentameric gH/gl complex. To differentiate the role of the pentameric gH/gl complex versus the functions of other ORFs in the 15-kb region in supporting viral growth in vivo, a series of recombinant viral strains were constructed and their ability to replicate in SCID-hu mice was tested. The mutations in the Towne and AD169 strains were repaired to restore their pentameric gH/gl complex and it was found that these changes did not rescue their inability to replicate in the SCID-hu mice. Subsequently four deletion viruses (D1, D2, D3, and D4) in the 15-kb region from the Toledo strain were created. It was demonstrated that D2 and D3 were able to grow in SCID-hu mice, while D1 and D4 were not viable. Interestingly, co-infection of the implant with the D1 and D4 viruses could compensate their respective growth defect in vivo. The results demonstrated that rescuing viral epithelial tropism is not sufficient to revert the attenuation phenotype of AD169 or Towne, and pathogenic genes are located in the segments missing in D1 and D4 viruses. J. Med. Virol. 88:1417-1426, 2016. © 2016 Wiley Periodicals, Inc.

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo.

The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

ORF7 of varicella-zoster virus is a neurotropic factor.

Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens.

Use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones.

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

Histone deacetylase 3, not histone deacetylase 2, interacts with the major immediate early locus of human cytomegalovirus.

Evidence suggests that genome chromatinization and the posttranslational modification of histones are involved in the regulation of viral gene expression, including the human cytomegalovirus (HCMV). We performed a ChIP-on-Chip assay to determine whether histone deacetylases (HDACs) interact with HCMV genomic DNA on a global level. Surprisingly, we found that HDAC3, but not HDAC2, interacts not only with the major immediate early (MIE) promoter but also with the entire MIE locus, suggesting a heterogeneous interaction of HDAC3 with HCMV DNA. The interaction of HDAC3 with the MIE region is related to inhibition of viral replication because HDAC3 inhibitors enhanced HCMV replication.

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2.

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.

Development of a gene capture method to rescue a large deletion mutant of human cytomegalovirus.

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that causes serious clinical illness in immunocompromised individuals. A major breakthrough in the progression of HCMV genetics studies is the development of bacterial artificial chromosome (BAC) clones of the viral genome. Recently, a luciferase reporter gene was inserted in the BAC clone (BAC(luc)) which facilitates monitoring of the virus growth both in vitro and in vivo. The virus made from the BAC(luc) grew with the similar kinetics as the wild-type strain indicating that the luciferase gene insertion does not interfere with the virus growth. Although the construction of the BAC clone has eased genetic studies of herpesviruses tremendously, there are still difficulties in cloning large DNA fragments of the virus to rescue mutations with large deletions. This paper describes a novel method termed "gene capture", which allows easier cloning of large pieces of DNA. As an application of this method, a 15-kb fragment that was deleted from the HCMV genome was rescued back into the viral genome. A mutant HCMV clone with the 15-kb region deletion was generated first using the lambda prophage recombination system in E. coli. Utilizing the new rescue method, the deleted fragment was then rescued in two steps: the 15-kb region was captured into a vector by homologous recombination; and the captured DNA fragment from the vector was inserted back into its native site in the mutant viral BAC by second homologous recombination. This method will be useful particularly for cloning large DNA fragments from any genome without introducing undesired mutations by traditional PCR-based approaches.

Cytotoxicity of dinitrotoluenes (2,4-dNT, 2,6-DNT ) to MCF-7 and MRC-5 cells.

DNTs are considered possibly carcinogenic to humans (Group 2B) because there is inadequate evidence in humans for carcinogenicity though there is sufficient evidence in experimental animals. In this study, MCF-7 (breast) and MRC-5 (lung) cells were exposed to a serial dilution of 2,4 and 2,6 DNTs (control, 1-500 ppm) in 96 well tissue culture plates. After various time intervals (24, 48, 72 and 96 hrs) the plates were washed, and 100microl fluorescein diacetate solution (10 microg/ml in PBS) was added column wise to each well, and incubated at 37 C for 30 - 60 min before reading the fluorescence with a spectrofluorometer at excitation and emission wavelengths of 485 and 538 nm respectively. Spectrofluorometeric readings were converted to percentages of cell survival. Regression analysis was conducted to determine the relationship between cell survival and exposed concentration. Linear equations derived from the regression analysis were used to calculate the LC50 values. Results indicated that 2,6 DNT was more toxic to breast cells; LC50 values were 445 and 292 ppm at 24 and 48 hours respectively compared to 2,4 DNT showing LC50 values of 570 and 407 ppm at 24 and 48 hours, respectively. No significant differences in toxicity existed between the two chemicals with regard to lung cells. Contrary to the above observation, 2,4 DNT was more toxic to breast cells; LC50 values were 407 and 238 ppm at 24 and 48 hours respectively compared to lung cells showing LC50 values of 527 and 402 ppm at 24 and 48 hours respectively. No significant difference existed for 2,6 DNT between the two cell lines. Lungs cells were more resistant to the two chemicals.