Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates

Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5′-3′-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5′ and 3′ untranslated regions (UTRs) of the viral genome. Furthermore, LSm1–7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV life cycle requirements and highly conserved host proteins of cellular mRNA decay. The previously described role of these proteins in the replication of 2 other positive-strand RNA viruses, the plant brome mosaic virus and the bacteriophage Qß, pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.     

Novel 3′ Proximal Replication Elements in Umbravirus Genomes

The 3′ untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3′UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3′ cap-independent translation enhancers (3′CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3′TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3′ terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed “Trio”, that are exclusively found in a subset of umbraviruses and are located just upstream of the 3′TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3′ terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3′CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.     

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTR_P162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5B_P162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.     

Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3′ UTR of turnip crinkle virus

The 3^′ untranslated region (3^′ UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined three-dimensional structure of a functional element in the 3^′ UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.     

Inhibition of SARS-CoV-2 Replication by a Small Interfering RNA Targeting the Leader Sequence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5′-untranslated region (5′-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options.     

Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5′ exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5′ RNA sequence as substrate (G_pppAG-RNA), leading to the formation of the 5′ RNA cap: G_0pppAG-RNA→^m7G_0pppAG-RNA (“cap-0”)→^m7G_0pppA_m2′-O-G-RNA (“cap-1”). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5′-^m7G_0pppA₁G₂U₃U₄G₅U₆U₇-3′), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2′-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.Several members of the Flavivirus genus from the Flaviviridae family are major human pathogens, such as the four serotypes of dengue virus (DENV1–4), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV). Recent large-scale DENV vaccine trials using a tetravalent formulation and three dose injections have shown only limited protection against the four DENV serotypes, and no specific antiviral drug has reached the market so far (1–3). The flavivirus genome consists of a (+)-sense single-stranded RNA of ∼11 kb with a type 1 cap structure, followed by the strictly conserved dinucleotide sequence “AG”: 5′-^m7G_pppA_m2′-O-G-3′ (4, 5). Addition of the cap moiety to the 5′ end of the viral genome is crucial for viral replication, because this structure ensures efficient production of viral polyproteins by the host translation machinery and protection against degradation by 5′-3′ exoribonucleases, and also because it conceals the triphosphorylated (or diphosphorylated) end from recognition by host cell innate immune sensors (6–9). Following (+)-strand RNA synthesis by the C-terminal RNA-dependent RNA polymerase (RdRp) domain of nonstructural protein 5 (NS5), cap formation in flaviviruses results from several sequential enzymatic reactions carried out by (i) the RNA triphosphatase activity of the NS3 protease-helicase that hydrolyzes the γ-phosphate group of the viral 5′ untranslated region (UTR), yielding a diphosphate RNA, (ii) a guanylyl-transferase activity proposed to reside in the methyltransferase (MTase) domain of NS5, which transfers a GMP molecule to the 5′-diphosphate RNA, and (iii) NS5-mediated sequential N-7- and 2′-O-methylations according to the following scheme: G_0pppAG-RNA→^m7G_0pppAG-RNA (“cap-0”)→^m7G_0pppA_m2′-O-G-RNA (“cap-1”) (5, 10–12).During flavivirus RNA replication, 5′-guanosine N-7-methylation is shown to be essential for translation of the viral polyprotein (13), whereas 2′-O-methylation on the penultimate A nucleotide conceals the viral genome from host immune sensors, notably RIG-I (14), MDA5 (15), and IFIT1 (16–18). Specifically, WNV carrying the E218A mutation in NS5 (E216A in DENV3 NS5) devoid of 2′-O (but not N-7) MTase activity was attenuated in wild-type but not Ifit1^−/− cells (16). Furthermore, the translation of JEV viral proteins was inhibited by IFIT1 through direct binding to the 5′-capped 2′-O-unmethylated mRNA (17). More recently, 2′-O-methylation at internal adenosines (but not at G, C, or U positions) by the flavivirus NS5 protein was demonstrated. The functional consequence of methylation at internal adenosines was an attenuation of viral RNA translation and replication (12). In vitro, the MTase domain of NS5 catalyzes these two enzymatic reactions with distinct requirements of RNA substrates and buffers: 5′-guanosine N-7 methyl transfer is optimal on a 211-nt segment of the 5′UTR at pH 6 and is inhibited by MgCl₂, whereas adenosine ribose 2′-O-methylation only requires a short RNA with “AG” as the first two RNA nucleotides and is maximal at pH 9–10 in the presence of Mg²⁺ ions. Thus, NS5 plays a crucial role both in virus replication and evasion of the host innate immune response, and therefore constitutes an attractive therapeutic target for antiviral drug and vaccine development (2, 19).Several crystal structures of flavivirus MTases have been reported either as free enzymes or bound to GTP (20), to the broad antiviral nucleoside analog ribavirin (21), to short cap analogs (22, 23), and to a capped-RNA octamer (24). Collectively, these structures uncovered a GTP binding site, the S-adenosyl-methionine (SAM) methyl-donor binding pocket, and a basic cleft at the protein surface that was proposed to accommodate the incoming RNA substrate. However, in the absence of a viral RNA in a catalytically meaningful position, the mechanism accounting for specific viral RNA methylation, including the structural basis for specific adenosine 2′-O-methylation, remains elusive. Moreover, the size of the RNA substrate that can be accommodated by the putative RNA binding cleft is also unknown, as well as any requirement for a specific RNA conformation. Determination of the structure of NS5 bound to a viral RNA would give valuable information to guide the design of specific NS5 inhibitors.To elucidate how the flavivirus RNA is recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein (DENV serotype 3), an authentic cap-0 viral RNA substrate (5′-^m7G_0pppA₁G₂U₃U₄G₅U₆U₇-3′), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. Together with mutagenesis data informed by the present structure, this work reveals a unique and specific interaction between the protein and viral RNA and provides the molecular basis for the methyl transfer reaction. Furthermore, despite a low sequence identity between the two proteins, the RNA recognition mode is reminiscent of how the human 2′-O-ribose methyltransferase CMTr1 binds mRNA for cap formation, suggesting that the viral methyltransferase might derive from its eukaryotic homolog.     

Poly(rC)-Binding Protein 1 Limits Hepatitis C Virus Virion Assembly and Secretion

The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5′ untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1’s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.     

A Novel Motif in the 3′-UTR of PRRSV-2 Is Critical for Viral Multiplication and Contributes to Enhanced Replication Ability of Highly Pathogenic or L1 PRRSV

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) with enhanced replication capability emerged in China and has become dominant epidemic strain since 2006. Up to now, the replication-regulated genes of PRRSV have not been fully clarified. Here, by swapping the genes or elements between HP-PRRSV and classical PRRSV based on infectious clones, NSP1, NSP2, NSP7, NSP9 and 3′-UTR are found to contribute to the high replication efficiency of HP-PRRSV. Further study revealed that mutations at positions 117th or 119th in the 3′-UTR are significantly related to replication efficiency, and the nucleotide at position 120th is critical for viral rescue. The motif composed by 117–120th nucleotides was quite conservative within each lineage of PRRSV; mutations in the motif of HP-PRRSV and currently epidemic lineage 1 (L1) PRRSV showed higher synthesis ability of viral negative genomic RNA, suggesting that those mutations were beneficial for viral replication. RNA structure analysis revealed that this motif maybe involved into a pseudoknot in the 3′-UTR. The results discovered a novel motif, 117–120th nucleotide in the 3′-UTR, that is critical for replication of PRRSV-2, and mutations in the motif contribute to the enhanced replicative ability of HP-PRRSV or L1 PRRSV. Our findings will help to understand the molecular basis of PRRSV replication and find the potential factors resulting in an epidemic strain of PRRSV.     

Brief Report: Measuring infectious SARS-CoV-2 in clinical samples reveals a higher viral titer:RNA ratio for Delta and Epsilon vs. Alpha variants

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.     

Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided.Virions of positive-strand RNA viruses contain genomic RNA of messenger sense. After infection, genomic RNA is released from the virions into the cytoplasm and translated to produce viral proteins, including viral RNA-dependent RNA polymerases and other replication-related proteins. These proteins are collectively called “replication proteins.” In eukaryotic positive-strand RNA viruses, replication proteins recruit genomic RNA to the cytoplasmic face of intracellular membranes to form replication complexes (1, 2). Negative-strand RNAs that are complementary to genomic RNAs are synthesized in the replication complexes, and then, using the negative-strand RNAs as templates, genomic RNA is copied and released into the cytoplasm. The recognition of template RNAs and their recruitment to the replication complexes are key processes in selective amplification of genomic RNA by positive-strand RNA viruses. In several positive-strand RNA viruses, cis-acting elements for replication-template selection have been identified, and, for some of them, it was demonstrated that replication proteins directly bind to these elements (3).Replication of tobacco mosaic virus (TMV), poliovirus, and many other positive-strand RNA viruses is cis-preferential: i.e., replication proteins recognize their own translation templates for replication (4–13). Because viral RNA replication is error-prone, it is important for viruses to selectively eliminate defective genomes. Template selection in cis is apparently advantageous in this regard because the genomes that encode replication proteins of lower performance are amplified less efficiently. Despite its importance in viral replication as well as evolution, little is known about how replication proteins select a template RNA in cis although it was proposed that requirement of nascent or newly synthesized replication proteins for replication and restricted diffusion or integrity of the proteins underlie the phenomenon (6).The genomic RNAs of positive-strand RNA viruses serve as templates for both translation and negative-strand RNA synthesis. During negative-strand RNA synthesis, viral RNA polymerases move along genomic RNA templates in a 3′-to-5′ direction. On the other hand, ribosomes synthesize viral proteins moving along the genomic RNA templates in a 5′-to-3′ direction. If these reactions take place on a single genomic RNA molecule at the same time, RNA polymerases and ribosomes collide, which results in the collapse of both reactions because these molecules cannot reverse direction or detach from the template RNA (14). Thus, positive-strand RNA viruses must clear ribosomes from the genomic RNA strands before negative-strand RNA synthesis occurs (15, 16).TMV belongs to the alpha-like virus superfamily of positive-strand RNA viruses. Its genome is a 5′-capped monopartite RNA and encodes at least four proteins, including the 5′ terminal 126-kDa protein, its translational read-through product of 183 kDa, a 30-kDa cell-to-cell movement protein, and a 17.5-kDa coat protein (17). The 126-kDa and 183-kDa proteins are replication proteins (18). The 126-kDa protein harbors a methyltransferase-like domain that is involved in RNA 5′ capping in its N-terminal region and a helicase-like domain in its C-terminal region. A region between these two domains is called the intervening region, or IR. The read-through part of the 183-kDa protein contains a polymerase-like domain (19). A deletion derivative of TMV RNA, named TMV126 RNA, that encodes the 126-kDa protein but not the 183-kDa protein can replicate when the 183-kDa protein is supplied in trans from a helper virus. However, TMV126 mutants that do not encode functional 126-kDa protein cannot replicate even if the wild-type 126-kDa and 183-kDa proteins are supplied in trans (8). This and other observations indicate that the 126-kDa protein functions primarily in cis (20, 21). The 5′ untranslated region (UTR) of TMV genomic RNA called Ω is ∼70 nucleotides (nt) in length, contains 12 CAA repeats, and is reported to have unusual tertiary structure with non-Watson–Crick base pairing (22, 23). The 5′ UTR of TMV RNA is a well-known translation enhancer (24, 25) and is essential for efficient virus multiplication (26). However, the role of the 5′ UTR in replication has been unclear, mainly due to the lack of experimental systems to separately evaluate translation of viral RNA and negative- and positive-stand RNA synthesis.To dissect the process that precedes the formation of the tobamovirus RNA replication complex on membranes, we previously developed an in vitro translation-replication system (27). Using an evacuolated tobacco protoplast extract (BYL) from which membranes were removed by centrifugation (membrane-depleted BYL, or mdBYL), we demonstrated that the replication proteins of tomato mosaic virus (ToMV), a close relative of TMV, bind ToMV RNA to form a ribonucleoprotein complex named premembrane-targeting complex (PMTC) in a translation-coupled manner (28). The PMTC is inactive in RNA synthesis but forms an active replication complex capable of synthesizing negative-strand and positive-strand RNA when it is mixed with membranes prepared from BYL. PMTC-like ribonucleoprotein (core-PMTC) is formed when a ToMV derivative that expresses the 126-kDa protein, but not the 183-kDa protein, is translated in mdBYL, which can form a replication complex when the 183-kDa protein and membranes are posttranslationally supplied (28). In the current study, we characterized tobamovirus PMTC and obtained results that provide insight into how the genomic RNA of TMV is selected as a template for replication preferentially in cis as well as how collisions between replication proteins and ribosomes are avoided.     

Single nucleotide polymorphism in RECQL and survival in resectable pancreatic adenocarcinoma

Background:

RECQL is a DNA helicase involved in DNA mismatch repair. The RECQL polymorphism, 3′ untranslated region (UTR) A159C, was previously associated with overall survival of patients with resectable pancreatic adenocarcinoma treated with neoadjuvant chemoradiation. In the present study, we examined RECQL for somatic mutations and other polymorphisms and compared these findings with the outcome in patients who received adjuvant or neoadjuvant chemoradiation. We hypothesized that RECQL (i) would be mutated in cancer, (ii) would have polymorphisms linked to the 3′UTR A159C and that either or both events would affect function. We also hypothesized that (iii) these changes would be associated with survival in both cohorts of patients.

Material and methods:

We sequenced RECQL''s 15 exons and surrounding sequences in paired blood and tumour DNA of 39 patients. The 3′UTR A159C genotype was determined in blood DNA samples from 176 patients with resectable pancreatic adenocarcinoma treated with adjuvant (53) or neoadjuvant (123) chemoradiation. Survival was calculated using the Kaplan–Meier method, with log rank comparisons between groups. The relative impact of genotype on time to overall survival was performed using the Cox proportional hazards model.

Results:

Somatic mutations were found in UTRs and intronic regions but not in exonic coding regions of the RECQL gene. Two single nucleotide polymorphisms (SNPs), located in introns 2 and 11, were found to be part of the same haplotype block as the RECQL A159C SNP and showed a similar association with overall survival. No short-term difference in survival between treatment strategies was found. We identified a subgroup of patients responsive to neoadjuvant therapy in which the 159 A allele conferred strikingly improved long-term survival.

Discussion:

The RECQL 3′UTR A159C SNP is not linked with other functional SNPs within RECQL but may function as a site for regulatory molecules. The mechanism of action needs to be clarified further.