Introduction Materials and Methods Results
The ESCRT Machinery is Involved in KSHV Assembly and Egress Justine Chiou; Lorenzo Gonzalez, PhD; Yan Yuan, PhD
Department of Microbiology, School of Dental Medicine, University of Pennsylvania
Introduction
Materials and Methods
Results
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the e8ologic agent for Kaposi’s sarcoma (KS), the most common malignant cancer associated with HIV infec8on1. Though KS lesions can affect different organ systems, 50% of AIDS pa8ents with KS manifest oral lesions, with 15% presen8ng in the oral cavity as the ini8al site2,3. Not only is oral KS common, it is also more aggressive and malignant than lesions occurring at other sites of the body, with pa8ents having a less than 10% 5-year survival rate4. In KS lesions, KSHV’s ly8c replica8on, virion release, and con8nual infec8on of fresh cells are crucial for viral tumorigenicity. Despite the importance of ly8c produc8on in viral pathogenesis, many processes of the KSHV ly8c cycle, including virion assembly and egress, remain poorly understood. This project sought to elucidate key proteins in the endosomal sor8ng complexes required for transport (ESCRT) pathway appropriated by KSHV.
Two approaches were employed to examine if the ESCRT pathway is involved in KSHV virion assembly. 1) shRNA (knockdown) method: • Knock down ESCRT pathway components (TSG101, ALIX-1, CHMP2A, and VPS4A) via len8viral shRNA transduc8on of BCBL-1 cells. • Measure encapsidated KSHV genomic DNA (gDNA) produc8on by real-8me PCR to evaluate the func8onal consequences of virion assembly. 2) Dominant nega8ve (overexpression) method: • Transfect BCBL-1 cells with plasmids expressing dominant nega8ve forms of ESCRT pathway components (TSG101, ALIX, CHMP1A, and VPS4A). • Effects assayed as above.
Although the ESCRT early ac8ng factors TSG101 and ALIX have been previously demonstrated to be important for HIV-1 envelopment, silencing of TSG101 and ALIX-1 expression by shRNA had non-significant effects on KSHV viral budding. In contrast, CHMP2A and VPS4A inhibi8on demonstrated reduc8on in KSHV gDNA produc8on, indica8ng impairment in viral assembly and egress. Via dominant nega8ve overexpression, TSG101 and ALIX similarly had minimal effect, while CHMP1A and VPS4A also reduced virion produc8on.
Ubiqui8n-mediated coordina8on of the vesicula8on process (adapted from Grabbe et al., 2011)
shRNA Knockdown
**P < 0.01 * P < 0.05
125 100 75
* **
50 25
1) KSHV acquires its membranous envelope by using host ESCRT components. 2) In preliminary screening, CHMP1A, CHMP2A, and VPS4A are essen8al for late phase KSHV assembly and egress, while 3) TSG101 and ALIX-1 are considered nonessen8al in the final stage of the KSHV life cycle. Though TSG101 and ALIX-1 are involved in viral Dominant Negative Overexpression budding of other enveloped viruses, KSHV may ****P < 0.0001 TSG101 use not-yet-iden8fied components to recruit *** P < 0.0002 ALIX 150 CHMP1A ESCRT to the viral budding site on endosomal VPS4A 125 membranes.
Clinical Significance
100 75 50 25
*** ****
**** ****
TS ble G 10 TS 1-1 G 10 12 A LI C H X-1 M P2 C H A -1 M P2 A VP -2 S4 A VP -1 S4 A -2
m ra
Figure 1. Knockdown of targeted ESCRT proteins via shRNA decreases virion producGon. Effect of shRNA k n o c k d o w n i s n o r m a l i z e d b y extracellular/ intracellular KSHV genome copy number. Each shRNA targets a specific sequence while scramble is a non-targe8ng control. C H M P 2 A a n d V P S 4 A s h R N A knockdown significantly decreased KSHV virion produc8on.
Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN
0
0
Sc
% Virion Production
% Virion Production
150
Conclusion
Figure 2. Overexpressing mutant forms of essenGal proteins in KSHV decreases virion producGon. Effect of dominant nega8ve (DN) overexpression with 2.5μg and 5μg of indicated plasmid and their respec8ve wild-type controls was quan8fied by KSHV DNA copy number as a func8onal consequences of virion produc8on. Mutant CHMP1A and VPS4A reduced KSHV assembly and egress in a dose-dependent manner.
Further study of KSHV assembly and egress may assist in the development of drugs that target specific proteins in the ESCRT pathway that inhibit the ability of KSHV to hijack its machinery for virion matura8on, thereby yielding addi8onal treatments for KSHV infec8on. References: 1. Shecy, K. Management of oral Kaposi’s sarcoma lesions on HIV-posi8ve pa8ent using highly ac8ve an8retroviral therapy: Case report and a review of the literature. Oral Oncology Extra 41, 226–229 (2005). 2. Green, T. L., Beckstead, J. H., Lozada-Nur, F., Silverman, S. & Hansen, L. S. Histopathologic spectrum of oral Kaposi's sarcoma. Oral Surg. Oral Med. Oral Pathol. 58, 306–314 (1984). 3. Nichols, C. M., Flaitz, C. M. & Hicks, M. J. Trea8ng Kaposi's lesions in the HIV-infected pa8ent. J Am Dent Assoc 124, 78–84 (1993). 4. Payne, S. F., Lemp, G. F. & Rutherford, G. W. Survival following diagnosis of Kaposi's sarcoma for AIDS pa8ents in San Francisco. J. Acquir. Immune Defic. Syndr. 3 Suppl 1, S14–7 (1990). Acknowledgements: Funded by the Penn Dental Medicine summer research program. Special thanks to Drs. Yan Yuan, Lorenzo Gonzalez, the en8re Yuan lab, the Vernon Brightman Research Society, and the PDM research commicee for their kind guidance and support.
Department of Microbiology, School of Dental Medicine, University of Pennsylvania
Introduction
Materials and Methods
Results
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the e8ologic agent for Kaposi’s sarcoma (KS), the most common malignant cancer associated with HIV infec8on1. Though KS lesions can affect different organ systems, 50% of AIDS pa8ents with KS manifest oral lesions, with 15% presen8ng in the oral cavity as the ini8al site2,3. Not only is oral KS common, it is also more aggressive and malignant than lesions occurring at other sites of the body, with pa8ents having a less than 10% 5-year survival rate4. In KS lesions, KSHV’s ly8c replica8on, virion release, and con8nual infec8on of fresh cells are crucial for viral tumorigenicity. Despite the importance of ly8c produc8on in viral pathogenesis, many processes of the KSHV ly8c cycle, including virion assembly and egress, remain poorly understood. This project sought to elucidate key proteins in the endosomal sor8ng complexes required for transport (ESCRT) pathway appropriated by KSHV.
Two approaches were employed to examine if the ESCRT pathway is involved in KSHV virion assembly. 1) shRNA (knockdown) method: • Knock down ESCRT pathway components (TSG101, ALIX-1, CHMP2A, and VPS4A) via len8viral shRNA transduc8on of BCBL-1 cells. • Measure encapsidated KSHV genomic DNA (gDNA) produc8on by real-8me PCR to evaluate the func8onal consequences of virion assembly. 2) Dominant nega8ve (overexpression) method: • Transfect BCBL-1 cells with plasmids expressing dominant nega8ve forms of ESCRT pathway components (TSG101, ALIX, CHMP1A, and VPS4A). • Effects assayed as above.
Although the ESCRT early ac8ng factors TSG101 and ALIX have been previously demonstrated to be important for HIV-1 envelopment, silencing of TSG101 and ALIX-1 expression by shRNA had non-significant effects on KSHV viral budding. In contrast, CHMP2A and VPS4A inhibi8on demonstrated reduc8on in KSHV gDNA produc8on, indica8ng impairment in viral assembly and egress. Via dominant nega8ve overexpression, TSG101 and ALIX similarly had minimal effect, while CHMP1A and VPS4A also reduced virion produc8on.
Ubiqui8n-mediated coordina8on of the vesicula8on process (adapted from Grabbe et al., 2011)
shRNA Knockdown
**P < 0.01 * P < 0.05
125 100 75
* **
50 25
1) KSHV acquires its membranous envelope by using host ESCRT components. 2) In preliminary screening, CHMP1A, CHMP2A, and VPS4A are essen8al for late phase KSHV assembly and egress, while 3) TSG101 and ALIX-1 are considered nonessen8al in the final stage of the KSHV life cycle. Though TSG101 and ALIX-1 are involved in viral Dominant Negative Overexpression budding of other enveloped viruses, KSHV may ****P < 0.0001 TSG101 use not-yet-iden8fied components to recruit *** P < 0.0002 ALIX 150 CHMP1A ESCRT to the viral budding site on endosomal VPS4A 125 membranes.
Clinical Significance
100 75 50 25
*** ****
**** ****
TS ble G 10 TS 1-1 G 10 12 A LI C H X-1 M P2 C H A -1 M P2 A VP -2 S4 A VP -1 S4 A -2
m ra
Figure 1. Knockdown of targeted ESCRT proteins via shRNA decreases virion producGon. Effect of shRNA k n o c k d o w n i s n o r m a l i z e d b y extracellular/ intracellular KSHV genome copy number. Each shRNA targets a specific sequence while scramble is a non-targe8ng control. C H M P 2 A a n d V P S 4 A s h R N A knockdown significantly decreased KSHV virion produc8on.
Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN Co 2. n 5 tro µg l 5 D µg N DN
0
0
Sc
% Virion Production
% Virion Production
150
Conclusion
Figure 2. Overexpressing mutant forms of essenGal proteins in KSHV decreases virion producGon. Effect of dominant nega8ve (DN) overexpression with 2.5μg and 5μg of indicated plasmid and their respec8ve wild-type controls was quan8fied by KSHV DNA copy number as a func8onal consequences of virion produc8on. Mutant CHMP1A and VPS4A reduced KSHV assembly and egress in a dose-dependent manner.
Further study of KSHV assembly and egress may assist in the development of drugs that target specific proteins in the ESCRT pathway that inhibit the ability of KSHV to hijack its machinery for virion matura8on, thereby yielding addi8onal treatments for KSHV infec8on. References: 1. Shecy, K. Management of oral Kaposi’s sarcoma lesions on HIV-posi8ve pa8ent using highly ac8ve an8retroviral therapy: Case report and a review of the literature. Oral Oncology Extra 41, 226–229 (2005). 2. Green, T. L., Beckstead, J. H., Lozada-Nur, F., Silverman, S. & Hansen, L. S. Histopathologic spectrum of oral Kaposi's sarcoma. Oral Surg. Oral Med. Oral Pathol. 58, 306–314 (1984). 3. Nichols, C. M., Flaitz, C. M. & Hicks, M. J. Trea8ng Kaposi's lesions in the HIV-infected pa8ent. J Am Dent Assoc 124, 78–84 (1993). 4. Payne, S. F., Lemp, G. F. & Rutherford, G. W. Survival following diagnosis of Kaposi's sarcoma for AIDS pa8ents in San Francisco. J. Acquir. Immune Defic. Syndr. 3 Suppl 1, S14–7 (1990). Acknowledgements: Funded by the Penn Dental Medicine summer research program. Special thanks to Drs. Yan Yuan, Lorenzo Gonzalez, the en8re Yuan lab, the Vernon Brightman Research Society, and the PDM research commicee for their kind guidance and support.
