Polyomaviridae Spring 2002 331
Antibody Prevalence for BK & JC Viruses Spring 2002 332
Polyoma Viruses General characteristics Papovaviridae: PA - papilloma; PO - polyoma; VA - vacuolating agent a. 45nm icosahedral viruses which contain a double-stranded, covalently closed circular genome of approximately 5 kb. b. Limited number of members (see table) 1. Family Papovaviridae, subfamily polyomaviruses 2. SV40 and polyomavirus the best characterized viruses of this group. c. Tend to have limited host range 1. Ex.: SV 40 infects rhesus monkeys but can also be forced to infect newborn hamsters, some cell cultures. Pathology: 1. Seldom cause tumors in their natural hosts 2. Often induce neoplastic cell transformation (cancer) in tissue culture or when introduced into experimental animals. 3. Cells infected by polyomaviruses can respond in one of two ways: a. Productive (lytic) infection: progeny virus produced which result in the death of the cells b. Non-productive (abortive) infection: interruption of the viral replication cycle, no infectious progeny produced Spring 2002 333
SV40 Genome Genome organization (see figure) a. The genome is split into two regions: 1. Early region: encoded genes are expressed in the early stages of virus infection -Early gene proteins are termed T antigens 2. Late region: encode genes expressed late in the virus replication cycle -Tend to be the viral structural proteins (VP1, VP2, & VP3) b. The DNA has a single origin of replication which is surrounded by non-translated regions containing cis acting sequences for controlling DNA replication and transcription. 1. Transcription proceeds bidirectionally from near the origin of replication -Early mrnas are transcribed from one strand of DNA -Late mrnas are transcribed from the other DNA strand. Spring 2002 334
SV 40 Genome Spring 2002 335
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Life Cycle a. Attachment to the host cell through VP1 MHC interaction b. Mechanism of entry is endocytosis c. Uncoating 1. SV40 is transported to the nucleus in vesicles, where they fuse with the outer nuclear membrane and deliver the virus particles into the perinuclear space. 2. Uncoating occurs in the nucleus, but the details of uncoating are unknown. d. Transcription of early mrnas 1. Carried out by cellular RNA Pol II 2. mrnas for the early gene products have a common 5'-terminus which maps 20-70 nt upstream of the AUG of the translational start. 3. Early promoter region: a. The promoter resemble "normal" eukaryotic promoters: i. -30 TATA box (Directs start point of transcription) ii. GC boxes located at -40 to - 130 (Sp 1 binding site) iii. 72bp repeat enhancers (binding sites for additional transcription factors). iv. pre mrnas undergo alternative splicing to produce mrnas for large and small T antigen a. The nuclear localized, host cell spliceosome complex is utilized to alternatively process the pre mrna into two forms. b. Splice site selection is controlled by: 1. Structural elements intrinsic to the pre-mrna itself 2. Trans acting factors can also influence the choice of introns to be spliced Spring 2002 337
e. Synthesis of T/t antigens 1. Obviously, both T/t antigens are translated from their respective mrnas in the cytoplasm 2. Large T antigen is relocalized to the nucleus where it functions to regulate early transcription and viral DNA replication. a. a 7 aa. nuclear localization signal is utilized. b. A small amount (<5%) of T antigen is localized to the plasma membrane where it is found in a glycosylated (but apparently Golgi independent pathway) 3. Small t antigen is partitioned between the nucleus and the cytoplasm a. Function: 1. Controls the accumulation of viral DNAs 2. Binds specifically to cellular proteins f. Large T antigen plays a multifunctional role in the viral replication cycle: 1. Large T as a negative regulator of early transcription: a. Large T antigen binds to the early SV40 promoter (centered around -75) and blocks RNA Pol II binding. b. Plays an important role in switching from early to late gene expression 2. Large T antigen as a regulator of DNA replication. 3. Large T induces ribosomal RNA synthesis and cellular DNA synthesis 4. Large T can "immortalize" primary cell cultures a. Can bind p53 (a tumor suppresser gene product) g. Viral DNA replication: A model system for eukaryotic chromosomal replication 1. Large T antigen activates DNA replication: a. T antigen binds in the vicinity of the DNA origin of replication Spring 2002 338
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b. T antigen has both ATPase and helicase activity 2. DNA replication model : a. Large T binding at the origin core 1. A 64 bp region is bound by Large T in the presence of cell factors a. The SV 40 origin overlaps the early mrna start site 2. Origin consists of a 15-20 bp AT rich region on the late site, a GC-rich palindromic center, and an inverted repeat of 14 bp on the early gene side - Binding of T antigen at origin allows helicase activity to unwind DNA to create a replication bubble. - Cellular DNA polymerases initiate bidirectional DNA synthesis -Synthesis at the replication forks proceeds bidirectionally on the circular viral genome, terminating about 180o away. 3. Replication is semidiscontinous: One strand is synthesized continuously (the advancing replication fork). The other strand is synthesized discontinuously by RNA-primed synthesis (Okazaki fragments) 4. Two loops of nascent dsdna are formed as replication intermediates which are topologically relaxed. But the template is negatively supercoiled. As replication proceeds, topoisomerase releases torsional strain as the DNA helix is unwound. 5. Termination and separation of daughter DNA molecules when the two replication forks meet. Spring 2002 340
Bidirectional DNA Replication Spring 2002 341
SV40 Origin of DNA Rep Spring 2002 342
SV40 DNA Initiation Spring 2002 343
h. Transcription of late mrnas begins after DNA replication has proceeded. 1. Large pre-mrna precursor molecules are produced that are subsequently processed to form cytoplasmic mrnas that are polyadenylated and spliced 2. Late promoter lacks TATA: a. The 72 bp repeat enhancer simulates late transcription in the presence of T antigen b. GC boxes also stimulate transcription 3. SV40 late mrnas are bicistronic: a. VP2 and VP3 from one mrna b. VP1 and agnoprotein are translated from another mrna i. Synthesis of late proteins 1. VP1, VP2, and VP3 are synthesized in the cytoplasm and transported to the nucleus where virion assembly takes place. j. Assembly and cell lysis 1. Viral DNA is first assembled with cellular histones H1, H2A, H2B, H3, and H4 (75S) 2. Viral structural proteins associate to form an immature nuclear provirion (240S) 3. H1 is removed during formation of mature extracellular virion. 4. 10 5 virus particles accumulate per cell resulting in death and cell lysis. Spring 2002 344
SV40 Structure 1. High resolution structure of SV40 has been solved to 3.8Åresolution a. 360 copies of the major coat protein VP1 (~40kD) b. VP2, VP3 are found in minor amounts and are not visible in the structure c. VP1 organized into 72 pentamers: i. 12 pentamers are found at the 5-fold rotation axes (vertex), surrounded by 5 pentamers ii. the remaining 60 pentamers do not lie on symmetry axes and are surrounded by 6 pentamers d. three kinds of pentamer-pentamer interactions: 1 three-fold and 2 types of two-fold 2. Structure of VP1: three modules a. N-terminal arm (~27 residues, 15 are disordered) i. extends into neighboring subunits within the pentamer b. an anti-parallel b-sandwich (~280 residues) i. similar to pico and plant virus coat proteins however strands are longer, have more extensive loops and have different shape ii. strands run parallel to the 5-fold axis iii. is the same structure in all environments iv. extensive interactions within subunits to generate pentamers pentamers can be found as basic building blocks c. a long C-terminal extension (~45 residues) i. deletion of C-terminal extension does not prevent formation of pentamers 3. Higher order structure: pentamer-pentamer contacts a. exclusively due to the invasion of neighboring pentamer C-terminal extensions b. a molecular clamp within the b-sandwich domain binds the C-terminal extension into the adjacent subunits c. the different subunits interact in slightly different environments to generate the overall structure Spring 2002 345
SV40 Structure Spring 2002 346
SV40 Structure Spring 2002 347
SV40 Transformation 1. Properties of Transformed Cells Transformed cells have lost the ability to regulate their own growth Growth of normal cells is regulated in cell culture by three factors 1. cell density 2. availability of growth factors 3. availability of solid surface to grow on Transformed cells overcome these limitations 2. SV40 transformation (also polyoma) a. ability to produce tumors in experimental animals in not normal. SV40 grows in monkey cells but transforms non-permissive hamster cells (no virus produced) b. although true oncogenes, the T antigens of polyomaviruses are not related to cellular oncogenes c. they are multifunctional proteins that share limited homology to cellular proteins in certain domains d. they have been studied by mutational analysis and by overexpression in tissue culture, additionally biochemical studies in vitro have examined the roles of these proteins in binding cellular proteins Spring 2002 348