Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

UNLABELLED: The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE: While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.

Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner.

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.

Human papillomavirus E1 helicase interacts with the WD repeat protein p80 to promote maintenance of the viral genome in keratinocytes.

Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.

Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases.

Bovine viral diarrhea virus (BVDV) is amongst the best-characterized members of the Flaviviridae, that includes the hepatitis C virus (HCV). The virally encoded RNA-dependent RNA polymerase (RdRp) plays a crucial role during replication and therefore represents an important target for the development of antiviral drugs. Here we studied biochemical mechanisms associated with the inhibition of BVDV RNA synthesis by 2'-hydroxyl, 3'-deoxynucleoside triphosphates (3'-dNTPs). All four nucleotide analogues are effectively incorporated and act as chain-terminators. However, relatively low, physiologically relevant concentrations of pyrophosphate (PPi) are sufficient to drive the reaction backwards, which results in primer unblocking and rescue of RNA synthesis. Metal ion requirements for nucleotide incorporation and pyrophosphorolysis are similar; the efficiency of both reactions is higher with Mn2+ as compared to Mg2+. Complexes containing chain-terminated primer strands are stable in the presence of heparin, which increases the probability that pyrophosphorolysis occurs before the enzyme can dissociate from its nucleic acid substrate. In contrast to the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT), the BVDV RdRp may not recruit NTP pools as PPi donors. Conversely, we found that the efficiency of primer unblocking is severely compromised in the presence of increasing concentrations of the NTP that is complementary to the next template position. These data suggest that the incoming NTP can access its designated binding site, which, in turn, prevents the catalytically competent complexation of PPi. The results of this study provide novel insights into mechanisms involved in pyrophosphorolysis associated with viral RdRps, and suggest that the excision reaction is likely to be an important parameter that can affect susceptibility to nucleotide analogue inhibitors directed against viral RdRps.

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1.

The bovine papillomavirus type 1 (BPV1) E2 protein binds as a dimer to the viral genome to promote its transcription, replication and maintenance in keratinocytes. Although BPV1 E2 dimerizes primarily through its DNA-binding domain, it was shown previously that its transactivation domain (TAD) can also dimerize in vitro through formation of a disulfide bond between cysteine 57 (C57) of adjacent monomers and of an ion pair between arginine 172 (R172) and aspartic acid 175 (D175). The function of this TAD dimerization interface in vivo remains unknown. Here, we report the effects of substituting C57, R172 and D175 by alanine on the transactivation activity of BPV E2 as well as on its ability to support viral DNA replication using a novel luciferase-based assay. Results for this mutational analysis suggest that the TAD dimerization interface is not essential for either process but may contribute to the DNA replication activity of BPV1 E2.

Molecular mechanisms of human papillomavirus-induced carcinogenesis.

Approximately 20% of all cancers are associated with infectious agents. Among them, human papillomaviruses (HPVs) are very common and are now recognized as the etiological agent of cervical cancer, the second most common cancer in women worldwide, and they are increasingly linked with other forms of dysplasia. Carcinogenesis is a complex and multistep process requiring the acquisition of several genetic and/or epigenetic alterations. HPV-induced neoplasia, however, is in part mediated by the intrinsic functions of the viral proteins. In order to replicate its genome, HPV modulates the cell cycle, while deploying mechanisms to escape the host immune response, cellular senescence and apoptosis. As such, HPV infection leads directly and indirectly to genomic instability, further favouring transforming genetic events and progression to malignancy. This review aims to summarize our current understanding of the molecular mechanisms exploited by HPV to induce neoplasia, with an emphasis on the role of the 2 viral oncoproteins E6 and E7. Greater understanding of the role of HPV proteins in these processes will ultimately aid in the development of antiviral therapies, as well as unravel general mechanisms of oncogenesis.

Pyrophosphorolytic excision of nonobligate chain terminators by hepatitis C virus NS5B polymerase.

Nonobligate chain terminators, such as 2'-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3'-hydroxyl group can be excised in the presence of pyrophosphate (PP(i)), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2'-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2'-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2'-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2'-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

High resolution footprinting of the hepatitis C virus polymerase NS5B in complex with RNA.

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

Control of template positioning during de novo initiation of RNA synthesis by the bovine viral diarrhea virus NS5B polymerase.

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.