Role of Middle T-Small T in the Lytic Cycle of Polyomavirus: Control of the Early-to-Late Transcriptional Switch and Viral DNA Replication

The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine ␤-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

Spink

Laimins

2005

“…Middle T and small T also play important roles in different aspects of polyomavirus infection (21,23,55). Defects in viral DNA replication and transcription, as well as defects in viral assembly, have been observed in different mutants of middle T and small T (1,7,8,22,38). Each of the viral early proteins also contributes to regulation of host cell function.…”

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confidence: 99%

Genetic Analysis of the Polyomavirus DnaJ Domain

Whalen

Jesus

Kean

et al. 2005

Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60 c-src and in transformation.Polyomavirus T antigens function both in replication of the virus and in transformation of the host cell. Large T is central to virus production as the initiator of viral DNA replication (20). Middle T and small T also play important roles in different aspects of polyomavirus infection (21,23,55). Defects in viral DNA replication and transcription, as well as defects in viral assembly, have been observed in different mutants of middle T and small T (1,7,8,22,38). Each of the viral early proteins also contributes to regulation of host cell function. Large T is able to immortalize primary cells (44), to block differentiation (37), and to provoke apoptosis (18, 48). These activities are mediated via association with the retinoblastoma susceptibility (Rb) family of tumor suppressors. Middle T, the major transforming protein, works through activation of cellular signaling pathways that are regulated by src-family tyrosine phosphorylation (15). Small T is able to promote cell cycle progression via association with protein phosphatase 2A (PP2A) (39).All three T antigens are produced by differential splicing of common primary transcripts (56). As a result, they have the identical N-terminal sequence of 79 amino acids that encompasses a DnaJ domain. DnaJ domains, consisting of approximately 70 amino acids, have a helical structure in which a conserved HPD motif is found between helix 2 and helix 3 (2, 13, 32, 43, 54). DnaJ domains, found in a broad range of proteins, function to stimulate ...

“…The repressor activities of transcription factors may participate in the early to late switch of transcription in the lytic cycles of Py and SV40 (49,91): when late gene expression is activated, repressor complexes might be disassociated from transcription factors and replaced by activator complexes containing histone acetyltransferase activities. This might be achieved by titration through replicating viral DNA (49,91,97) or by direct modification of transcription factors or repressor complexes through middle T-and/or small t-mediated signaling (13). Similar control may be exerted upon DNA replication during the viral life cycle.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Polyomavirus ori- Dependent DNA Replication by mSin3B

Xie

Folk

2002

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) oridependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.The mammalian Sin3 (mSin3) transcriptional corepressor is associated with many distinct protein complexes, among which, the mSin3/histone deacetylases (HDAC)/RbAp48 complex is predominant (reviewed in references 4 and 59). This complex is recruited by unliganded nuclear hormone receptors through N-CoR/SMRT (nuclear receptor corepressor/silencing mediator for retinoid and thyroid receptors) (2,32,34,45,57,102), the methylated CpG-binding protein MeCP2 (40, 58), Mad/ Max and Mxi/Max heterodimers (71), p53 (56), and other proteins (reviewed in reference 15) to repress the transcription of specific targets.At least two forms of mSin3B are found in mammalian cells due to alternative mRNA splicing: the full-length mSin3B (mSin3B LF ) containing four paired amphipathic ␣-helix (PAH) and a short form mSin3B (mSin3B SF ) containing only the first two PAH domains and lacking the HDAC1/2-interacting domain (2). HDAC1 and -2 interact with mSin3A (or -B) protein through domains within the C terminus, starting at PAH3 (2, 92), and HDAC7 interacts with PAH1 of mSin3 (42), whereas HDAC5, as well as HDAC7, indirectly interacts with mSin3 through N-CoR/SMRT (37,42). N-CoR/SMRT also acts as a corepressor in an mSin3-HDAC complex (2,32,34,45,57,102). The L59P substitution in PAH1 impairs interaction with N-CoR/SMRT and partially reduces corepressor activity (2). Not all active Sin3 complexes require either HDAC activity or even the presence of HDACs (2,41,45,92,100).HDAC-dependent p53-mediated transcriptional repression requires mSin3 (56, 103). The p53 protein also represses in vitro replication of simian virus 40 (SV40) DNA and polyomavirus (Py) DNA (19,55,82,86) and nuclear DNA replication in the Xenopus egg extracts (14). Whether or not mSin3 is required for p53 mediated repression of DNA replication is not known.HDAC complexes recruited by pRB to deacetylate histones and nonhistone proteins important for transcription also contain mSin3 (8, 46, 50, 51). pRB's control of cell cycle progression and DNA replication depends, at least in part, upon repressing transcription of E2F-regulated genes through both HDAC-dependent and -independent mechanisms (31, 46). That control of DNA replication by pRB might involve mechanisms besides transcriptional repression is suggested by observations including: (i) interaction of pRB with the replication-licensing factor MCM7 inhibits DNA replication in vitro (...