The Rose lab's current research areas include:
- Investigating the role of the oropharyngeal environment in the transmission and pathogenesis of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8)
- Cellular and viral receptors facilitating KSHV entry during virus infection
- The latency-associated animal models of human disease
- Characterization of rhadinovirus genes involved in latency and lytic replication
- Consensus Degenerate Hybrid Oligonucleotide Primers for Gene and Pathogen Discovery (CODEHOP)
- Virus diagnostics
Investigating the role of the oropharyngeal environment in the transmission and pathogenesis of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8)
In 2010, Rose and his colleagues received a program project grant from the National Institutes Health (NIH) to study how KSHV infection leads to Kaposi's sarcoma (KS). Rose and others on the research team - including Drs. Serge Barcy and Soren Gantt of Seattle Children's, Dr. Michael Lagunoff from the University of Washington and Dr. Corey Casper from Fred Hutchinson Cancer Research Center - played key roles in the discovery and characterization of KSHV and related viruses in non-human primates, and in the role of the oral environment in the acquisition and transmission of the virus.
Furthermore, the team was at the forefront of characterizing the role of the immune response in regulating KSHV infection and transmission. KSHV is often transmitted via saliva and oral KS is a common consequence of KSHV in HIV-infected people.
In this program project, the Rose lab's research focuses on how the virus is transmitted from cell to cell and disseminates from the initial site of infection in the oral cavity to distal sites in lymphoid, epithelial and endothelial tissues. Lagunoff studies how KSHV induces KS tumor growth, while Barcy and Gantt study the immune responses that control oral and systemic KSHV infection, and how HIV impairs these responses.
Together, these projects study the clinical relevance of the findings by collaborating with Casper at the Uganda Cancer Institute/Hutchinson Center Cancer Alliance, a joint venture with the Fred Hutchinson Cancer Research Center and the Uganda Cancer Institute. The alliance's mandate is to facilitate research, training and clinical care in infection-related cancers, and to provide fiscal and administrative management to collaborative programs. The Alliance is based at the Uganda Cancer Institute in Kampala, where the epidemiology, pathophysiology, natural history and treatment of infection-related cancers has been studied since 2004.
Due to the high prevalence of KSHV infection, the pediatric and adult populations in Uganda are highly vulnerable to the development and spread of KSHV-associated malignancies, which have become health problems of enormous proportions. The World Health Organization has determined that KS is the most common cancer in the general population throughout East Africa and is also the malignancy with the highest mortality. The long-term goal of the program project is to increase the understanding of the biological basis of KS, in order to develop novel strategies for prevention and treatment.
More recently, Rose obtained an administrative supplement to the program project enabling a collaboration with Drs. Damien Chaussabel and Peter Linsley of the Benaroya Research Institute to conduct a genome-wide comprehensive analysis of global KSHV, cellular and immune gene expression in their complement of unique tissue samples from Uganda.
- Garrigues, HJ, Rubinchikova, YE, Rose TM (2014). KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells. Virol 452:75-85.
- Gutierrez KD, Morris VA, Wu D, Barcy S, Lagunoff M (2013). Ets-1 is required for the activation of VEGFR3 during latent Kaposi's sarcoma-associated herpesvirusinfection of endothelial cells. J Virol. 87 (12):6758-68.
- Dimaio TA, Lagunoff M (2012). KSHV Induction of Angiogenic and LymphangiogenicPhenotypes. Front Microbiol. 3:102.
- Gantt S, Kakuru A, Wald A, Walusansa V, Corey L, Casper C, Orem J. (2010). Clinical presentation and outcome of epidemic Kaposi sarcoma in Ugandan children. Pediatr Blood Cancer. 54 (5):670-4
- Barcy S, De Rosa SC, Vieira J, Diem K, Ikoma M, Casper C, Corey L (2008). Gammadelta+ T cells involvement in viral immune control of chronic human herpesvirus 8infection. J Immunol. 180 (5):3417-25. PubMed PMID: 18292568.
Cellular and Viral Receptors Facilitating KSHV Entry During Virus Infection
TRecent studies have highlighted the importance of the oral environment for the biological life cycle and transmission of Kaposi's sarcoma-associated herpesvirus (KSHV), and its association with the AIDS-related malignancy Kaposi's sarcoma (KS). Saliva has been identified as the only significant source of transmissible infectious virus, and oral epithelial cells (keratinocytes) are believed to be the primary source of infectious virus in vivo. Additional studies suggest that the oropharynx is an important site for the initial acquisition of a KSHV infection. Little is known, however, regarding the biology of KSHV in oral keratinocytes at any of the levels of virus recognition by epithelial cell receptors; biological response to infection of epithelial cells; or production and characterization of infectious virions from epithelial cells.
The Rose lab's is working to identify and characterize the viral and cellular factors involved in KSHV entry and infection of oral keratinocytes. In 1999, Rose, in association with collaborators at the University of Washington, sequenced the KSHV glycoprotein B, a major constituent of the KSHV virion envelope. They identified an "RGD" motif in glycoprotein B which is known to bind to members of the integrin family of cell surface receptors, including integrin alphaV/beta3. Rose and colleagues obtained a U.S. patent for the use of glycoprotein B peptides, polynucleotides and antibodies for the diagnosis of KSHV infection and eliciting immune responses in KSHV vaccines.
Dr. Jacques Garrigues, a longtime member of the Rose lab team, has continued the work on the role of KSHV glycoprotein B in KSHV entry. Garrigues determined that a peptide containing the KSHV glycoprotein B RGD motif specifically binds to integrin alphaV/beta 3 and function blocking antibodies inhibit adhesion of cells to the RGD peptide. Using affinity-purified integrins and confocal microscopy, he demonstrated that alphaV/beta3 integrin bound to the glycoprotein B RGD motif and to KSHV virions, demonstrating direct receptor-ligand interactions. Specific alphaV/beta3 antagonists, including cyclic and dicyclic RGD peptides and alphaV/beta3 function-blocking antibodies, strongly inhibited KSHV infection.
These studies demonstrated that integrin alphaV/beta3 was a cellular receptor mediating both cell adhesion and entry of KSHV into target cells through binding the virion-associated glycoprotein B RGD motif.
- Rose, T.M., Strand, K.B., and Bosch, M.L. (1999). Glycoprotein B of the RFHV/KSHV subfamily of herpesviruses. United States Patent #6,015,565
- Garrigues HJ, Rubinchikova YE, Dipersio CM, Rose TM (2008). Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor. J Virol 82 (3):1570-1580. PMCID: PMC2224453.
The Latency-Associated Animal Models of Human Disease
In 1997, Rose and colleagues at the University of Washington discovered a new herpesvirus, retroperitoneal fibromatosis herpesvirus (RFHV), in a macaque tumor that is similar to Kaposi's sarcoma. The Rose lab has characterized RFHV by sequencing critical regions of the viral genome, including the divergent locus B; ORF73 - the latency-associated nuclear antigen (LANA); and ORF59 - the DNA polymerase processivity factor. More recently, the complete genome sequence of RFHVMn, from a pig-tailed macaque (M. nemestrina), was determined by next-gen sequencing of DNA from an RF tumor.
The sequence was obtained from an archived frozen RF tumor carrying ~3 RFHV genomes per cell by Illumina-based sequencing of the complete complement of DNA present in the tumor, reducing the DNA reads from the macaque genome bioinformatically and performing a de novo assembly of the RFHV genome sequence. Comparison of the RFHVMn and KSHV genomes revealed close genetic and genomic similarities, suggesting a common biology, life cycle and pathology. These similarities demonstrate that RFHV infection in macaques is a close animal model of KSHV infection in humans.
In 2000, Rose and others determined that two homologs of KSHV existed in macaques and several other non-human primate species, constituting two distinct rhadinovirus lineages in Old World primates. KSHV and RFHV grouped within the RV1 rhadinovirus lineage with other closely related homologs of KSHV. The second RV2 rhadinovirus lineage consisted of rhesus rhadinovirus (RRV), pig-tailed rhadinovirus (MneRV2) and other closely related rhadinoviruses. Non-human primates are infected with viruses from both lineages. An RV2 rhadinovirus infecting humans has not yet been discovered.
The Rose lab has shown that the macaque RV1 rhadinoviruses are present in the spindeloid tumor cells of the macaque retroperitoneal fibromatosis tumors, while the macaque RV2 rhadinoviruses are present in the lymphoid tumor cells of B-cell and T-cell lymphomas of macaques with SIV-associated AIDS. The Rose lab is characterizing the macaque RV1 (RFHVMn and RFHVMm) and RV2 (RRV and MneRV2) rhadinoviruses from rhesus (Mm) and pig-tailed macaques (Mn) to better understand their biology and differences in virus life cycle, transmission and pathology. The goal is to use sequence and functional comparisons with KSHV to determine the roles of critical viral genes to better understand KSHV and its role in the development of KS.
- Bruce, A.G., Ryan, J.T., Thomas, M.J., Peng, X., Grundhoff, A, Tsai, C-C, Rose, T.M. (2013) Next-generation sequence analysis of the genome of RFHVMn, the macaque homolog of KSHV, from a KS-like tumor of a pig-tailed macaque. J. Virol. 87, 13676-13693.
- Ryan, J.T. and Rose, T.M. (2013) Development of whole-virus multiplex Luminex-based serological assays for diagnosis of infections of KSHV/HHV8 homologs in macaques. Clin Vaccine Immunol. 20, 409-419.
- Bruce AG, Bielefeldt-Ohmann H, Barcy S, Bakke AM, Lewis P, Tsai CC, Murnane RD, and Rose TM (2012). Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related Lymphomas. PLoS Pathog 8 (10):e1002962
- Philipp-Staheli J, Marquardt T, Thouless ME, Bruce AG, Grant RF, Tsai CC, Rose TM (2006). Genetic variability of the envelope gene of Type D simian retrovirus-2 (SRV-2) subtypes associated with SAIDS-related retroperitoneal fibromatosis in different macaque species. Virol J 3:11.
- Bielefeldt-Ohmann H, Barouch DH, Bakke AM, Bruce AG, Durning M, Grant R, Letvin NL, Ryan JT, Schmidt A, Thouless ME, Rose TM (2005). Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque. (Macaca mulatta). Vet Pathol 42 (3):391-396.
- Bruce AG, Bakke AM, Thouless ME, Rose TM (2005). Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons. Virol J 2:2.
- Rose TM, Ryan JT, Schultz ER, Raden BW, Tsai CC (2003). Analysis of 4.3 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi's sarcoma-associated herpesvirus. J Virol 77 (9):5084-5097.
- Schultz ER, Rankin GW, Jr., Blanc MP, Raden BW, Tsai CC, Rose TM (2000). Characterization of two divergent lineages of macaque rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus. J Virol 74 (10):4919-4928.
- Rose TM, Strand KB, Schultz ER, Schaefer G, Rankin GW, Jr., Thouless ME, Tsai CC, Bosch ML (1997). Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species. J Virol 71 (5):4138-4144.
Characterization of Rhadinovirus Genes Involved in Latency and Lytic Replication
ORF73 latency-associated nuclear antigen (LANA)
The Rose lab cloned and sequenced the ORF 73 LANA homologs of the pig-tailed macaque RV1 and RV2 rhadinoviruses, RFHVMn and MneRV2, since pig-tailed macaques are the major macaque species in the WaNPRC. Several differences were observed between the RV1 and RV2 LANA homologs, including a large internal repeat region that was present in the LANAs of both HHV8 and RFHV but absent in the RV2 LANA. The repeat regions of both HHV8 and RFHV were characterized by repetitive reiterations of glutamic acid residues. The RRV and MneRV2 LANA homologs were significantly shorter and lacked the repeat region.
A comparison of the RFHV and HHV8 LANA sequences showed a low but significant sequence conservation throughout the N and C terminal regions (~30%) and the presence of conserved chromosome binding and nuclear localization signals. We determined that the monoclonal LN53 anti-KSHV LANA antibody reacted with the RFHVMn LANA. Using this antibody we showed that the spindeloid tumor cells in the macaque retroperitoneal fibromatosis tumor expressed LANA which correlated with the quantitative PCR data showing that the tumors contained high levels of RFHV DNA (~2-4 viral genomes/cell). This data strongly supported an etiological association of RFHV with the retroperitoneal fibromatosis tumors. Analysis of the nuclear localization signal of KSHV LANA revealed a strong sequence and functional conservation with RFHV LANA. We determined that the nuclear localization signal was bifunctional, allowing the LANA proteins to be imported into the nucleus using both the classical and non-classical nuclear import pathways. The multifunctional nuclear localization signal may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.
ORF59 DNA polymerase processivity factor
We have cloned and expressed the ORF 59 homologs of the RV1 and RV2 rhadinoviruses from the chimpanzee and three species of macaques. We found that the ORF59 homologs of the RV1 and RV2 rhadinoviruses are highly conserved with distinct phylogenetic clustering within the two rhadinovirus lineages. A rabbit antiserum was developed against a conserved sequence in the macaque RV2 ORF 59 sequences. This antiserum was used to show the presence of ORF59 positive infected cells within the differentiating layer of the epidermis. This strongly supports the idea that the differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses.
ORF50 replication transactivator (RTA)
We have analyzed the promoters for the RTA homologs in the rhesus rhadinovirus (RRV) and Kaposi's sarcoma-associated herpesvirus (KSHV). We identified cells that are not permissive for RRV replication and recapitulate the latent KSHV infection and reactivation processes. We identified a critical Sp1 element in the RRV RTA promoter that was conserved in the KSHV RTA promoter. These studies showed that while the outcome of KSHV infection was determined by LANA inhibition of the RTA promoter activity, the outcome of RRV infection was determined by host factors, such as Sp1.
- DeMaster LK and Rose TM (2014). A critical Sp1 element in the rhesus rhadinovirus (RRV) Rta promoter confers high-level activity that correlates with cellular permissivity for viral replication. Virol 448:196-209.
- Cherezova L, Burnside KL, and Rose, TM (2011). Conservation of Complex Nuclear Localization Signals Utilizing Classical and Non-Classical Nuclear Import Pathways in LANA Homologs of KSHV and RFHV. PLoS ONE 6:e18920.
- Bruce AG, Bakke AM, Gravett CA, DeMaster LK, Bielefeldt-Ohmann H, Burnside KL, and Rose TM (2009). The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques. Virol J 6:205.
- Bruce AG, Bakke AM, Bielefeldt-Ohmann H, Ryan JT, Thouless ME, Tsai CC, Rose TM (2006). High levels of retroperitoneal fibromatosis (RF)-associated herpesvirus in RF lesions in macaques are associated with ORF73 LANA expression in spindleoid tumour cells. J Gen Virol 87 (Pt 12):3529-3538.
- Bruce AG, Thouless ME, Tsai CC, Rose TM (2006). RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells. Virology 354 (1):103-115.
CODEHOP - Consensus Degenerate Hybrid Oligonucleotide Primers for Gene and Pathogen Discovery
The Rose lab developed a novel technique using consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the identification of distantly related genes. We have developed an interactive software program and website for the design of CODEHOP PCR primers for the identification of distantly related genes (iCODEHOP). We are working closely with the Washington National Primate Research Center to identify new pathogens infecting primates maintained at the center, and are developing CODEHOP PCR assays to detect novel primate virus species. We have used this technique to discover the macaque herpesviruses described above, as well as to identify other novel retroviruses and herpesviruses.
We are developing CODEHOP assays with broad specificity to detect both known and unknown viruses within different virus families. This approach enables the lab tests to detect low levels of virus while preserving the ability to detect known, unknown and mutated members of a virus family. CODEHOP assays have been developed to detect known and novel members of the papillomavirus, herpesvirus, adenovirus, paramyxovirus and influenzavirus families.
- Boyce R, Chilana P, Rose TM (2009). iCODEHOP: a new interactive program for designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply aligned protein sequences. Nucleic Acids Res. 37: W222-8.
- Staheli JP, Ryan JT, Bruce AG, Boyce R, and Rose TM (2009). Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) for the detection of novel viruses in non-human primates. Methods 49:32-41. PMCID: PMC2751581.
- Rose TM (2005). CODEHOP-mediated PCR - a powerful technique for the identification and characterization of viral genomes. Virol J 2:20.
- Rose TM, Henikoff JG, Henikoff S (2003). CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res 31 (13):3763-3766.
- Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998). Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26 (7):1628-1635.
- VanDevanter DR, Warrener P, Bennett L, Schultz ER, Coulter S, Garber RL, Rose TM (1996). Detection and analysis of diverse herpesviral species by consensus primer PCR. J Clin Microbiol 34 (7):1666-1671.
The Rose lab is working closely with Redmond, Washington-based Micronics, Inc. to develop point-of-care microfluidics-based diagnostic tests for both paramyxovirus and influenza virus families of respiratory viruses. These tests use nucleic acid amplification and are fast, portable and affordable enough to be utilized at point of care. The paramyxovirus assay can detect and differentiate the major paramyxovirus types, including respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and parainfluenzaviruses 1-4 (PIV1-4) in a single assay. The influenza virus assay is robust and can be used to detect both known and emerging influenza strains. This approach has the special utility of being able to detect unknown influenza strains that have pandemic capabilities, like the recent H1N1 "swine flu" and the current highly pathogenic H7N9 avian influenza strain.
- Staheli JP, Ryan JT, Bruce AG, Boyce R, and Rose TM (2009). Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) for the detection of novel viruses in non-human primates. Methods 49:32-41.