Single-cell analysis reveals host S phase drives large T antigen expression during BK polyomavirus infection.

BK polyomavirus (BKPyV) is a major cause of kidney transplant failure, for which there are no antivirals. The current model is that BKPyV expresses TAg (large T antigen) early during infection, promoting cells to enter S phase where the viral DNA can access the host replication machinery. Here, we performed a single-cell analysis of viral TAg expression throughout the cell cycle to reveal that robust TAg expression required replication of the host DNA first.

Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV).

BK Polyomavirus Requires the Mismatch Repair Pathway for DNA Damage Response Activation.

BK polyomavirus (PyV) infects the genitourinary tract of >90% of the adult population. Immunosuppression increases the risk of viral reactivation, making BKPyV a leading cause of graft failure in kidney transplant recipients. Polyomaviruses have a small double-stranded DNA (dsDNA) genome that requires host replication machinery to amplify the viral genome.

Binding of a viral IRES to the 40S subunit occurs in two successive steps mediated by eS25.

The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S-IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex.

BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase.

BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication.

Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper protein (HBZ) is expressed in all cases of ATL and is directly associated with virus pathogenicity. The two isoforms of the HBZ protein are synthesized from antisense messenger RNAs (mRNAs) that are either spliced (sHBZ) or unspliced (usHBZ) versions of the HBZ transcript.

Noncanonical Translation Initiation in Eukaryotes.

The vast majority of eukaryotic messenger RNAs (mRNAs) initiate translation through a canonical, cap-dependent mechanism requiring a free 5' end and 5' cap and several initiation factors to form a translationally active ribosome. Stresses such as hypoxia, apoptosis, starvation, and viral infection down-regulate cap-dependent translation during which alternative mechanisms of translation initiation prevail to express proteins required to cope with the stress, or to produce viral proteins. The diversity of noncanonical initiation mechanisms encompasses a broad range of strategies and cellular cofactors.

Translational Control in Virus-Infected Cells.

As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability.

Foxp1 Negatively Regulates T Follicular Helper Cell Differentiation and Germinal Center Responses by Controlling Cell Migration and CTLA-4.

T follicular helper (Tfh) cells play an essential role in the formation of germinal centers (GC) and generation of high-affinity Abs. The homing of activated CD4 T cells into B cell follicles and the involvement of key costimulatory and coinhibitory molecules are critical in controlling both the initiation and the magnitude of GC responses. Meanwhile, studies have shown that a high number of single clone B cells leads to intraclonal competition, which inhibits the generation of high-affinity Abs.

Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection.

The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection.

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

Background: There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs.

Cap-Independent Translational Control of Carcinogenesis.

Translational regulation has been shown to play an important role in cancer and tumor progression. Despite this fact, the role of translational control in cancer is an understudied and under appreciated field, most likely due to the technological hurdles and paucity of methods available to establish that changes in protein levels are due to translational regulation. Tumors are subjected to many adverse stress conditions such as hypoxia or starvation.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25).

Hu antigen R (HuR) is a positive regulator of the RNA-binding proteins TDP-43 and FUS/TLS: implications for amyotrophic lateral sclerosis.

Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators.

The 5' untranslated region of the human T-cell lymphotropic virus type 1 mRNA enables cap-independent translation initiation.

Unlabelled: The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5'-untranslated region (5'UTR) of the HTLV-1 full-length mRNA harbors an IRES.

Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection.

Ribosomal protein S25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting.

During viral infection or cellular stress, cap-dependent translation is shut down. Proteins that are synthesized under these conditions use alternative mechanisms to initiate translation. This study demonstrates that at least two alternative translation initiation routes, internal ribosome entry site (IRES) initiation and ribosome shunting, rely on ribosomal protein S25 (RPS25).

Tricks an IRES uses to enslave ribosomes.

In eukaryotes, mRNAs are primarily translated through a cap-dependent mechanism whereby initiation factors recruit the 40S ribosomal subunit to a cap structure at the 5' end of the mRNA. However, some viral and cellular messages initiate protein synthesis without a cap. They use a structured RNA element termed an internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit.

So you want to know if your message has an IRES?

Transcriptional regulation of gene expression has been widely studied. More recently, there has been increasing appreciation of the role that translational regulation plays in gene expression, resulting in a number of new fields engaging in translational studies. Regulation of protein synthesis is critical for cell growth, development, and survival, and is primarily controlled at the initiation step.

rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells.

How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A).

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites.

Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared.

Mechanism of translation initiation by Dicistroviridae IGR IRESs.

The Dicistroviridae is a growing virus family characterized by a dicistronic genome, wherein each open reading frame (ORF) is translated from an independent internal ribosome entry site (IRES). The 5' IRES that translates the first open reading frame (ORF1) is similar to the picornaviral IRESs. However the second IRES, referred to as the intergenic region (IGR) IRES, - translates ORF2 by and uses an unusual mechanism of initiating protein synthesis.

RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs.

Most eukaryotic mRNAs are translated using a cap-dependent mechanism of translation. However, approximately 10% of mammalian mRNAs initiate translation using a cap-independent mechanism that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) located in the 5' untranslated region.