eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

Peach latent mosaic viroid (PLMVd) is a small, single-stranded, circular RNA pathogen that infects Prunus persica trees. As with all other known viroids, the PLMVd genome does not encode any proteins. Consequently, it must interact with host cellular factors in order to ensure its life cycle. With the objective of identifying cellular proteins that interact with PLMVd, Northwestern hybridizations were performed using partially purified peach leaf extracts. Mass spectrometric analysis of the detected RNA-protein complexes led to the identification of six putative RNA-binding proteins. One of these was found to be elongation factor 1-alpha (eEF1A), and because of its known involvement in the replication and translation of various RNA viruses, further characterizations were performed. Initially, the existence of this interaction received support from an experiment that immunoprecipitated the eEF1A from a crude extract of infected peach leaves, coupled with reverse transcription-PCR detection of the PLMVd. Subsequently, eEF1A interaction with PLMVd strands of both polarities was confirmed in vitro by electrophoresis mobility shift assays, fluorescence spectroscopy, and the prediction of an altered PLMVd RNase mapping profile in the presence of the protein. The potential contribution of eEF1A to the molecular biology of PLMVd, including for viroid replication, is discussed.Viroids are small (250 to 400 nucleotides [nt]), singlestranded, circular pathogenic RNAs that infect higher plants, frequently causing significant losses in agriculture. In infected cells, viroids replicate in a DNA-independent manner via a rolling circle mechanism that follows either a symmetric or an asymmetric mode. In the symmetric mode, the infecting circular monomer, which is assigned a plus [(ϩ)] polarity by convention, is replicated into linear concatemeric minus [(Ϫ)] strands which are then spliced and ligated, yielding minusstrand circular monomers. By using the latter RNA molecules as templates, the same three steps are then repeated in order to produce the progeny. In contrast, in the asymmetric mode, the linear concatemeric (Ϫ) strands serve directly as the template for the synthesis of the linear concatemeric (ϩ) strands.There are 34 different viroid species known to belong to either the Pospiviroidae or Avsunviroidae family. Members of the Pospiviroidae family share a conserved central region, have a nuclear localization, and replicate via an asymmetric rolling circle mechanism. Conversely, the members of the Avsunviroidae are localized in the chloroplasts and replicate

Section: Discussionsupporting

confidence: 90%

Identification of Proteins fromPrunus persicaThat Interact with Peach Latent Mosaic Viroid

Dubé

Bisaillon

Perreault

2009

“…5I to K). Interestingly, a domain similar to the TD-NEM motif involved in non-CRM1-mediated protein nuclear export (30) and present in the CR3 domain appears to be important for this interaction (Fig. 5K).…”

Section: Discussionmentioning

confidence: 98%

“…Interestingly, the region of aa 70 to 84 contains a sequence (70-DAGIRNLQDCLL-81) which shows similarity to the consensus motif of the transcription-dependent nuclear export motif (TD-NEM motif) (DxGxxDxxL) (Fig. 5K, top) (30). The TD-NEM motif mediates the nuclear export of proteins independently of the classical CRM1/nuclear export signal (NES) system and requires ongoing RNA polymerase II (Pol II)-mediated transcription.…”

Section: Hpv38 E7 Disrupts Actin Stress Fibersmentioning

confidence: 99%

“…The TD-NEM motif mediates the nuclear export of proteins independently of the classical CRM1/nuclear export signal (NES) system and requires ongoing RNA polymerase II (Pol II)-mediated transcription. In this process, eEF1A plays a major role in nuclear exclusion by binding to the TD-NEM motif (30). We reasoned that the likely TD-NEM motif present in HPV38 E7 could mediate its interaction with eEF1A.…”

Section: Hpv38 E7 Disrupts Actin Stress Fibersmentioning

confidence: 99%

See 1 more Smart Citation

Cutaneous Human Papillomavirus Type 38 E7 Regulates Actin Cytoskeleton Structure for Increasing Cell Proliferation through CK2 and the Eukaryotic Elongation Factor 1A

Yue

Shukla

Accardi

et al. 2011

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.Human papillomaviruses (HPVs) are double-stranded small DNA viruses, comprising more than 100 members, that are the causative agents of several human diseases, including cancers (25). According to their tropisms, HPVs are subdivided into mucosal and cutaneous types. High-risk mucosal HPV types, such as HPV type 16 (HPV16) and HPV18, are etiological agents of cervical cancer and other anogenital cancers (25). Their ability to induce cellular immortalization and transformation is attributed primarily to the viral oncoproteins E6 and E7 (20,25). While E6 prevents apoptosis by inducing the degradation of the tumor suppressor p53 through the proteasome system, E7 disrupts cell cycle regulation by inactivating pRb (20,25). In addition, HPV16 E6 and E7 alter several other cellular signaling pathways by interacting with a number of cellular proteins, enhancing their carcinogenic properties (20,38,39,41).A large subgroup of cutaneous HPV types belonging to the beta genus of the HPV phylogenetic tree has been proposed to be involved in the development of nonmelanoma skin cancer (NMSC), since they were isolated for the first time from patients suffering from a rare autosomal recessive genetic disorder called epidermodysplasia verruciformis (EV), characterized by susceptibility to beta HPV infection and NMSC development at sun-exposed anatomical regions (7). Subsequent epidemiological studies have also provided lines of evidence for a possible oncogenic role of beta HPV types in non-EV patients, including the healthy population and immunocompromised individuals, e.g., organ transplant recipients (7, 51).Our group has previously reported t...

“…Furthermore, the export of PABP1 from the nucleus has been linked to both ongoing transcription and mRNA export from the nucleus (53,64). NSs shuts down host transcription and could thereby arrest nuclear export of PABP1.…”

Section: Discussionmentioning

confidence: 99%

Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

Copeland

Altamura

Deusen

et al. 2013

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.