**2. Viral gene functional studies**

84 Bacterial Artificial Chromosomes

sequence, or introduce new sequences without removing any of the existing sequences (gene removal or insertion). In addition BACs are also used to place nucleotide substitution through selection/counterselection strategy, and to conduct effective gap repair cloning of

Genes expressed from BACs mirror endogenous gene expression far more accurately than other cloning systems. The large size of BACs help to minimize site of integration effects, a phenomenon which has been defined as endogenous sequences (such as gene coding regions and distal regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes (Adamson, Jackson et al. 2011) in gene cloning technology. The larger sized BAC constructs contain enhancers and locus control regions, which leads to more accurate gene expression *in vivo* (Townes, Lingrel et al. 1985; Jones, Monks et al. 1995). 1995). The human genome BACs consist of the full gene structure, including untranslated regions, exons and introns, alternative promoters and splice sites and microRNA coding sequences. RNAs such as RNA splicing or microRNAs play very important role in gene regulation (Jackson and Standard 2007). Therefore the human genome BACs will ensure full mRNA processing and splicing when genes are transcribed, and produce the full complement of protein isoforms once mRNAs are translated. BACs can be transfected and expressed in mammalian cell lines although transfection efficiency and copy numbers are

BACs also have a number of disadvantages. A construct containing a large genomic fragment is likely to contain non-related genes that may lead to indirect, non-specific gene expression and unanticipated changes in the cell phenotype; Secondly, compared to plasmids or other gene expression vectors, the generation and screening of recombinant BAC constructs can be time-consuming and labor-intensive. Also, the oversized BAC DNA constructs are more easily sheared and degraded during manipulation before transfection; and some random recombination events may occur, for example, LoxP sites may lead to random Cre-mediated recombination (Semprini, Troup et al. 2007). Finally, repeating homologous sequences in some BACs constructs may undergo intramolecular rearrangements, which reduce the recombination efficiency and increase the rate of false-

low (Magin-Lachmann, Kotzamanis et al. 2004; Sparwasser and Eberl 2007).

positive clones in some selection/counter-selection approaches (Narayanan 2008).

Overall, BACs have numerous advantages when compared to conventional plasmids. They protect the gene from site of integration effects and produce accurate regulation of transcription and translation. However, the large size results in technical difficulties when handling them as well as the potential non-specific gene expression. Therefore the application of BACs as a gene expression model system should be careful considered based

**1.4 Application of BACs: Genomic sequencing, genomic imprinting, transgenic mice,** 

There is increasing interest in the application of BAC technology in genomic research. High throughput determination of gains and losses of genetic material using high resolution BAC

any target site of interest (Adamson, Jackson et al. 2011).

**1.3 Advantages and disadvantages of BACs** 

on the pros and cons previously described.

**vaccine development, and gene therapy** 

## **2.1 Many human and animal herpesviruses genomes have been cloned as BACs**

Human herpesviruses are the second leading cause of human viral disease. Therefore the utilization of human herpesvirus BACs to study viral gene function (Warden, Tang et al. 2011) has become more and more common. The herpesviruses are a family of DNA viruses which contain large and complex genomes. Genetic control and management of recombinant viruses have been notoriously difficult. The development of herpesvirus BACs have facilitated generation of recombinant viruses and subsequent studies of the biology and pathogenesis of herpesviruses (Knipe, Batterson et al. 1981; Zhou and Roizman 2005). Table 1 shows the human herpesviruses which have been cloned as BACs, including Herpes simples virus type 1 [(HSV-1 or human herpesvirus (HHV-1)], varicella-zoster virus (VZV or HHV-3), human cytomegalovirus (HCMV or HHV-5), Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) (Feederle, Bartlett et al. 2010; Warden, Tang et al. 2011). In general, BAC clones are relatively easy to make for alpha- and beta herpes viruses than gamma herpes viruses. This is due to the fact that DNA can only persistently stay in bacterial cells when it has a prokaryotic replicon. When a BACs flanked by specific Herpesvirus genomic sequences were introduced into infected cells to trigger homologous recombination. The great efficiency was achieved in alpha- and beta herpes viruses because lytic cellular systems are available, but was difficult for gamma herpesviruses (Delecluse, Hilsendegen et al. 1998; Delecluse, Kost et al. 2001; Zhou, Zhang et al. 2002; Kanda, Yajima et al. 2004; Chen, Li et al. 2007). In addition to BAC-based human herpesvirus studies, BACbased other animal herpesviruses are also currently available. These include murine cytomegalovirus 68 (MHV-68), murine gammaherpesvirus (mCMV), rhesus cytomegalovirus (rhCMV), rhesus rhadinovirus (RRV), pseudorabies virus (PrV), herpesvirus saimiri (HVS), Marek's disease virus (MDV), bovine herpesvirus type 1 (BHV-1), equine herpesvirus type 1 (EHV-1), feline herpesvirus (FHV-1), guinea pig cytomegalovirus (GPCMV), Koi herpesvirus (KHV) and turkery herpesvirus (HVT) (Feederle, Bartlett et al. 2010; Warden, Tang et al. 2011).


**type synonym subfamily biological function and application reference** 

Generates a replication-proficient but packaging-deficient HSV-1 genome

Generates a recombinant HSV-2 BAC with the deletion of the HSV-2

glycoprotein D (gD), elicites an HSV-2 specific antibody response, serves as the

transfected with VZV BAC DNA show cytopathic effect, and viruses can spread to neighboring cells.

basis for novel HSV-2 vaccine

Human embryonic lung cells

Luciferase VZV BAC generates recombinant VZV variants, eases subsequent viral growth kinetic analysis both *in vitro* MeWo cells and

The mini-F transposition technique optimizes, repairs or restructures BACs, facilitates the development of gene

Genetic analysis of all EBV functions, generation of attenuated EBV strains for vaccine design, development of viral vectors for human gene therapy.

Generates a self-recombining BAC containing 172-kb of the EBV genome; provides proof that EBNA-3B is not essential for EBV-mediated B-cell growth transformation *in vitro*.

SCID-hu mice *in vivo*.

therapy or vaccine vectors.

production.

(Saeki, Ichikawa et al. 1998; Stavropoulos and Strathdee

1998; Horsburgh, Hubinette et al. 1999)

(Meseda, Schmeisser et al. 2004)

(Nagaike, Mori et al. 2004)

(Zhang, Rowe et al. 2007)

(Wussow, Fickenscher et al. 2009)

(Delecluse, Hilsendegen et al. 1998; Kanda, Yajima et al. 2004)

(Chen, Divisconte et al. 2005)

(152-kb HSV-1) for genetic manipulation as research tools or

vectors in gene therapy.

HHV-1

HHV-2

HHV-3

(VZV)

HHV-4 Epstein-Barr

virus (EBV), lymphoc ryptovirus (LCV)

Varicella zoster virus

Herpes simplex virus-1 (HSV-1)

Herpes simplex virus-2 (HSV-2) α (Alpha)

α

α

γ (Gamma)


Table 1. List of available BACs for human HSV

## **2.2 vGPCR-mediated angiogenesis through activation of p38 and STAT3 in KSHV infected cells using KSHV BACs**

The molecular mechanism whereby viral G protein-coupled receptor (vGPCR) signaling regulates vascular endothelial growth factor (VEGF) expression in Kaposi sarcoma (KS) formation remains somewhat undefined. mECK36 cells, generated by transfection of mice bone marrow endothelial cells with KSHV bacterial artificial chromosome (KSHVBac36), have been reported to be angiogenic, tumorigenic, and suitable for demonstrating a nonredundant role for vGPCR in KSHV-mediated tumorigenesis (Mutlu, Cavallin et al. 2007). In our previous report (Liu 2010), we utilized mECK36, the cells composed of wild-type KSHVBac36 or the cells without vGPCR, namely vGPCR-null KSHVBac36 mutant, to dissect the molecular mechanisms of VEGF secretion induced by vGPCR in the context of KSHV infection. The mice bone marrow endothelial cells (mEC) were obtained from Balb/C An Ncr-nu mice (NCI, Bethesda, MD) bone marrow. Mice femurs were flushed twice with phosphate-buffered saline (PBS), and the elutes were incubated in Dulbecco's modified Eagle's medium (DMEM) media plus 30% fetal borine serum (FBS) (Gemini Bioproducts, Calabasas, CA), endothelial growth factor (EGF) 0.2 mg/mL (Sigma, St. Louis, MO), endothelial cell growth factor supplement (ECGS) 0.2 mg/mL (Sigma), heparin 1.2 mg/L (Sigma), insulin transferrine selenium (Invitrogen, Carlsbad, CA), penicilin-streptomicin 1% (Invitrogen), and BME vitamin (VWR Scientific, Rochester, NY). KSHVBac36 was constructed by inserting a full-length recombinant KSHV genome into a bacterial artificial chromosome, KSHVBac36 was transfected into mEC cells to generate mECK36 cells using lipofectamine 2000 (Invitrogen) and selected with hygromycin-B. The cells were then grown in the absence of hygromycin to negatively select cells and therefore generate mECK36-KSHV-Null cells, which lost the KSHV episome (KSHV episome was measured by GFP marker). Next, KSHVBac36 construct was retransfected into mECK36-KSHV-Null cells to generate BBac36. Finally, the genotypic markers of vGPCR were knocked out from KSHVBac36 by transposon mutagenesis to generate ORF74/vGPCR deletion mutant and stably transfected into mECK36-KSHV-Null cells to create BΔvGPCR cells in the presence of hygromycin selection. We found (Liu 2010) that vGPCR activates VEGF transcription via p38 MAPK and STAT3 in mECK36 and mECK36-derived cell models. In addition, we also found that in cells containing KSHV genome, STAT3 is tyrosinephosphorylated and translocated into the nucleus, transactivating the target VEGF gene by binding to the specific DNA element TT (N4–5) AA in a vGPCR-dependent manner. Moreover, treatment of mECK36-derived cells with AG490 or a dominant negative STAT3 DNA vector showed strong inhibitory effects on vGPCR-induced VEGF promoter activity. In addition, vGPCR can up-regulate STAT3 mRNA levels. Together, our findings show that vGPCR plays a nonredundant role in STAT3 activation in KSHV infected cells, and this activation plays an important role in the connection of the viral oncogene vGPCR and VEGF up-regulation. Our results indicate that vGPCR has a broad signaling activating capacity in the context of KSHV infection and suggest that the STAT3 pathway could be a good target for preventing KSHV-mediated angiogenesis in KS.

#### **2.3 Genetic determinants of virus tropism genes using BACs**

Many cell types, including endothelial cells (ECs), myeloid lineage cells, and smooth muscle cells are permissive cells for HCMV persistent replication and latency (Jarvis and Nelson 2007). During acute infection of CMV in immune-compromised patients, a number of cell types, such as ECs, various leukocytes, epithelial cells, hepatocytes, smooth muscle cells, and fibroblasts,

The molecular mechanism whereby viral G protein-coupled receptor (vGPCR) signaling regulates vascular endothelial growth factor (VEGF) expression in Kaposi sarcoma (KS) formation remains somewhat undefined. mECK36 cells, generated by transfection of mice bone marrow endothelial cells with KSHV bacterial artificial chromosome (KSHVBac36), have been reported to be angiogenic, tumorigenic, and suitable for demonstrating a nonredundant role for vGPCR in KSHV-mediated tumorigenesis (Mutlu, Cavallin et al. 2007). In our previous report (Liu 2010), we utilized mECK36, the cells composed of wild-type KSHVBac36 or the cells without vGPCR, namely vGPCR-null KSHVBac36 mutant, to dissect the molecular mechanisms of VEGF secretion induced by vGPCR in the context of KSHV infection. The mice bone marrow endothelial cells (mEC) were obtained from Balb/C An Ncr-nu mice (NCI, Bethesda, MD) bone marrow. Mice femurs were flushed twice with phosphate-buffered saline (PBS), and the elutes were incubated in Dulbecco's modified Eagle's medium (DMEM) media plus 30% fetal borine serum (FBS) (Gemini Bioproducts, Calabasas, CA), endothelial growth factor (EGF) 0.2 mg/mL (Sigma, St. Louis, MO), endothelial cell growth factor supplement (ECGS) 0.2 mg/mL (Sigma), heparin 1.2 mg/L (Sigma), insulin transferrine selenium (Invitrogen, Carlsbad, CA), penicilin-streptomicin 1% (Invitrogen), and BME vitamin (VWR Scientific, Rochester, NY). KSHVBac36 was constructed by inserting a full-length recombinant KSHV genome into a bacterial artificial chromosome, KSHVBac36 was transfected into mEC cells to generate mECK36 cells using lipofectamine 2000 (Invitrogen) and selected with hygromycin-B. The cells were then grown in the absence of hygromycin to negatively select cells and therefore generate mECK36-KSHV-Null cells, which lost the KSHV episome (KSHV episome was measured by GFP marker). Next, KSHVBac36 construct was retransfected into mECK36-KSHV-Null cells to generate BBac36. Finally, the genotypic markers of vGPCR were knocked out from KSHVBac36 by transposon mutagenesis to generate ORF74/vGPCR deletion mutant and stably transfected into mECK36-KSHV-Null cells to create BΔvGPCR cells in the presence of hygromycin selection. We found (Liu 2010) that vGPCR activates VEGF transcription via p38 MAPK and STAT3 in mECK36 and mECK36-derived cell models. In addition, we also found that in cells containing KSHV genome, STAT3 is tyrosinephosphorylated and translocated into the nucleus, transactivating the target VEGF gene by binding to the specific DNA element TT (N4–5) AA in a vGPCR-dependent manner. Moreover, treatment of mECK36-derived cells with AG490 or a dominant negative STAT3 DNA vector showed strong inhibitory effects on vGPCR-induced VEGF promoter activity. In addition, vGPCR can up-regulate STAT3 mRNA levels. Together, our findings show that vGPCR plays a nonredundant role in STAT3 activation in KSHV infected cells, and this activation plays an important role in the connection of the viral oncogene vGPCR and VEGF up-regulation. Our results indicate that vGPCR has a broad signaling activating capacity in the context of KSHV infection and suggest that the STAT3 pathway could be a good target for

**2.2 vGPCR-mediated angiogenesis through activation of p38 and STAT3 in KSHV** 

**infected cells using KSHV BACs** 

preventing KSHV-mediated angiogenesis in KS.

**2.3 Genetic determinants of virus tropism genes using BACs** 

Many cell types, including endothelial cells (ECs), myeloid lineage cells, and smooth muscle cells are permissive cells for HCMV persistent replication and latency (Jarvis and Nelson 2007). During acute infection of CMV in immune-compromised patients, a number of cell types, such as ECs, various leukocytes, epithelial cells, hepatocytes, smooth muscle cells, and fibroblasts, can be infected because of uncontrolled replication of viruses (Howell, Miller et al. 1979; Myerson, Hackman et al. 1984; Gnann, Ahlmen et al. 1988; Wiley and Nelson 1988; Dankner, McCutchan et al. 1990; Sinzger, Grefte et al. 1995; Read, Zhang et al. 1999; Bissinger, Sinzger et al. 2002). ECs appear to play a critical role in the process of HCMV persistent active infection and maintenance within the host, which is controlled by genetic determinants. Previous studies observed that HCMV strains differed in their ability to infect ECs, which are called EC tropism (MacCormac and Grundy 1999; Sinzger, Schmidt et al. 1999; Kahl, Siegel-Axel et al. 2000). The research on EC tropism has been strengthened by the availability of genetically stable CMV BACs and subsequent mutagenesis of these BACs (Brune, Menard et al. 2001; Scrivano, Sinzger et al. 2011). The switch of cell tropism in different cell types after alternate replication might direct infection from one cell type to the other.

The typical model for tropism is the difference in cell tropism of virus released from EC and fibroblasts. Supernatants from infected human foreskin fibroblasts (HFF) showed a higher ability to infect EC than EC-derived supernatants (Scrivano, Sinzger et al. 2011). Scrivano et al (Scrivano, Sinzger et al. 2011) using mutagenesis of the BAC-cloned HCMV strain TB40/E (TB40-BAC4) found that ECs release a virus progeny of unEC-tropic (not EC-tropic), and retain a progeny of highly EC-tropic; while HFF release both EC-tropic and non EC-tropic virus progeny, HFF progeny is composed of both EC-tropic and non EC-tropic virus populations. The biochemical basis for this phenomenon is due to a different level of gH/gL/pUL(128,130,131A) complex in virions (Scrivano, Sinzger et al. 2011). The CMV EC tropism has been characterized by a "genomic tropism island" composed of three open reading frames (ORFs): UL128, UL130, and UL131A. The region of these genes is important for EC tropism (Hahn, Revello et al. 2004; Scrivano, Sinzger et al. 2011). EC-tropic population most likely is a population with a high gH/gL/pUL(128,130,131A) content. UL128, UL130, and UL131A are required for replication of HCMV in HUVECs (Hahn, Revello et al. 2004). EC tropism for HCMV is highly dependent on the roles of pUL128, pUL130, and pUL131A (Jarvis and Nelson 2007) in virions. EC-tropism produced by an EC-tropic progeny released by HFF, can be depleted with antibodies directed against pUL131A. They propose that the difference in cell tropism of virus released from EC and fibroblasts is caused by a sorting process. EC strongly and specifically retain EC-tropic viruses through the gH/gL/pUL(128,130,131A) complex. Thus, the levels of gH/gL/pUL(128,130,131A) complexes could define whether a particle is EC-tropic or not. A disulfide-linked complex between gH/gL glycoproteins is required for viral entry and fusion. The gH/gL exists in two distinct forms, one composed of pUL128, pUL130, and pUL131A. The pUL128 and pUL130 proteins are linked with gH/gL; pUL131A is required for infection of ECs. The second distinct form is composed of gO alone; the gO protein is linked with gH/gL and is required for replication in fibroblasts. The gH/gL/pUL128/pUL130/pUL131A unit in virions is mandatory for access into ECs which are pH-dependent. Whereas the gH/gL/gO unit in virions are mandatory for access into fibroblasts which are also pH-independent (Jarvis and Nelson 2007). Recently, results from Wang et al showed (Wang, Yu et al. 2007) that HCMV progenies derived from epithelial cells and fibroblasts are also different. It seems the propensity of cells to release viruses plays a crucial role in the establishment of infection and transfer of viruses to new hosts or the fetus.

In addition to HCMV, EBV also works as a cell type-tropic virus. Hutt-Fletcher et al (Hutt-Fletcher 2007) has established the paradigm that epithelial cells produce a EBV virus progeny with high levels of gH/gL/gp42 complexes, facilitating B-cell infection. B-cells in turn, generate virus progeny with low levels of gH/gL/gp42 complexes which efficiently infect epithelial cells, but not B cells. To some extent, this relative switch of cell tropism after alternate replication in epithelial and B-cells directs infection from one cell type to the other.
