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The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

['Jorge Vera-Otarola', 'Laboratorio De Virología Molecular', 'Instituto Milenio De Inmunología E Inmunoterapia', 'Departamento De Enfermedades Infecciosas E Inmunología Pediátrica', 'Centro De Investigaciones Médicas', 'Escuela De Medicina', 'Pontificia Universidad Católica De Chile', 'Santiago', 'Unidad De Virología Aplicada', 'Dirección De Investigación Y Doctorados De La Escuela De Medicina']

Date: 2021-10

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

Andes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is unique among other members of the Hantaviridae family of viruses because of its ability to spread from person to person. The molecular mechanisms driving ANDV protein synthesis remain poorly understood. A previous report showed that translation of the Small segment mRNA (SmRNA) of ANDV relied on both the 5’cap and the 3’untranslated region (UTR) of the SmRNA. In this new study, we further characterize the mechanism by which the 5’ and 3’end of the SmRNA interact to assure viral protein synthesis. We establish that the viral nucleocapsid protein N and the cellular protein hMex3A participate in the process. These observations indicated that both viral and cellular proteins regulate viral gene expression during ANDV infection by enabling the viral mRNA to establish a non-covalent 5’-3’end interaction.

Funding: The work was supported by the Agencia Nacional de Investigacion y Desarrollo (ANID), Gobierno de Chile though the Iniciativa Cientifica Milenio (ICM), Instituto Milenio de Inmunología e Inmunoterapia (P09/016-F; ICN09_016), Programa de Investigación Asociativa PIA-ACT1408 to MLL, and FONDECYT 11150611 to JV-O, and by the Centre National de la Recherche Scientifique (CNRS, France), through the Laboratoire International Associé (LIA) program granted to MLL. EC-V conducted this research in partial fulfillment of the requirements for a Ph.D., Doctorado en Ciencias Biológicas mención Microbiología y Genética Molecular, Microbiología, Facultad de Biología, Pontificia Universidad Católica de Chile, funded by FONDECYT doctoral fellowships 21090539 and 24121224. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we were interested in evaluating if the ANDV-N could replace the function of eIF4F, as described for other orthohantavirus [ 12 ]. Also, we sought to further understanding how translation of the ANDV SmRNA overcomes the need for a poly(A) tail. Consistent with earlier reports from other orthohantavirus N proteins [ 12 , 26 ], we show that the ANDV-N stimulates mRNA translation in a 5’cap dependent fashion. However, in contrast to the N of other orthohantavirus [ 12 ], the ANDV-N could not substitute all functions of eIF4F. Data also confirm previous reports showing that the 3’UTR of the ANDV SmRNA plays an essential role during viral mRNA translation [ 27 ]. Furthermore, we report that the human RNA-binding protein Mex3A (hMex3A) binds to the ANDV SmRNA 3’UTR, promoting translation of the SmRNA. The ANDV-N and hMex3A additively stimulate translation from the virus-like SmRNA in cells. However, results show that the ANDV-N and the hMex3A do not interact. Nonetheless, both proteins independently interact with eIF4G. Thus, this study provides novel insights regarding the role of cellular and viral proteins in supporting ANDV SmRNA poly(A)-independent translation.

Eukaryotic mRNA translation comprises four stages initiation, elongation, termination, and ribosome recycling [ 13 – 16 ]. Translational control is exerted mainly at the initiation stage [ 14 , 15 ]. Translation initiation involves the eukaryotic initiation factor (eIF) 4F recognizing the mRNAs 5’cap, recruitment of the 40S ribosomal subunit, 5’-3’scanning of the mRNA until encountering the initiation codon, and the recruitment of the 60S ribosomal subunit [ 15 – 17 ]. Translation initiation ends with the 80S ribosome assembled and positioned at the initiation codon [ 15 – 17 ]. Eukaryotic mRNAs feature a 5’cap, and most have a 3’poly(A) tail [ 18 – 21 ]. The 5’cap and the 3’poly(A) tail establish a non-covalent interaction (closed-loop) that promotes efficient mRNA translation initiation, translation re-initiation through ribosome recycling, and enhanced mRNA stability [ 20 , 22 , 23 ]. For most eukaryotic mRNAs, the 5’-3’end interaction is mediated by the association of the capped-bound eIF4F with the poly(A)-bound poly(A)-binding protein (PABP) [ 20 , 24 , 25 ]. The heterotrimeric eIF4F protein complex comprises the cap-binding protein eIF4E, the RNA-dependent ATPase/RNA helicase eIF4A, and the high-molecular-weight scaffolding protein eIF4G [ 15 – 17 ]. Thus, the non-covalent mRNA circle is closed by eIF4G, which simultaneously interacts with the mRNA 5’capped-bound eIF4E and the 3’poly (A)-bound PABP [ 20 , 24 ]. In cells, assembly of the eIF4F complex and establishing the 5’cap-3’poly(A) interaction are tightly regulated processes [ 13 , 20 ].

The ANDV genome consists of three negative polarity single-stranded RNA segments, large (L), medium (M), and small (S), packed into helical nucleocapsids [ 4 ]. Transcription of the genomic RNA segments yields the 5’-7-methylguanosine (m 7 GTP) capped L, M, and S messenger RNAs (mRNAs). The mRNAs of orthohantavirus acquire their 5’cap- through a cap-snatching mechanism occurring in cytoplasmic processing bodies (P bodies) [ 5 ]. Interestingly, unlike most cellular mRNAs, the ANDV SmRNA and the LmRNA lack 3’poly(A) tails [ 6 ]. The LmRNA encodes a viral RNA-dependent RNA polymerase required for viral RNA transcription and replication [ 7 , 8 ]. The MmRNA encodes the glycoprotein precursor, which is cotranslationally processed, generating Gc and Gn, the two viral envelope glycoproteins [ 9 ]. The SmRNA encodes for the nucleocapsid protein (N) and the nonstructural S segment (NSs) protein [ 10 , 11 ]. Interestingly, the N-protein of other orthohantavirus participates in viral mRNA translation [ 12 ].

The Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of the Bunyavirales order, is the etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in Argentina and Chile [ 1 ]. ANDV mainly infects humans through aerosolized excreta and secretions from infected long-tailed pygmy rice rat (Oligoryzomys longicaudatus). A unique feature of ANDV is that its infection can also occur through person-to-person transmission [ 2 , 3 ].

Results

The ANDV-N protein stimulates translation of an ANDV-like mRNA in cells Next, we wondered whether the ANDV-N protein could stimulate the translation of the virus-like SmRNA in cells. For this, HEK 293T cells were transfected with a plasmid encoding a His-tagged ANDV-N protein (ANDV His-N). Forty-eight hours after, cells were transfected with the cap-N-RNA-3’UTR or the cap-N-RNA-poly(A) RNAs together with a capped and polyadenylated Renilla luciferase (RLuc) encoding mRNA (used as a control for RNA transfection efficiency). In the cap-N-RNA-poly(A) RNA, the ANDV SmRNA 3’UTR was substituted by a poly(A) tail [27]. Six hours post-RNA transfection, cells were lysed, and luciferase activities were determined and normalized to the RLuc control. Expression of the ANDV His-N protein was monitored by western blotting, using an anti-His antibody. In these assays, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (Fig 3A). FLuc activity was normalized to RLuc activity (FLuc/RLuc ratio), and results are presented as relative translation efficiency (RTA). The FLuc/RLuc ratios obtained for the cap-N-RNA-3’UTR and the cap-N-RNA-poly(A) RNAs alone were set to 100%. Results showed that in cells, expression of the ANDV His-N protein stimulated FLuc synthesis from the cap-N-RNA-3’UTR mRNA in a concentration-dependent manner, without impacting the translation of the cap-N-RNA-poly(A) mRNA (Fig 3B). These results suggest that in cells, translation stimulation of the virus-like RNA induced by the ANDV His-N protein depends on the presence of the 3’UTR of viral SmRNA. PPT PowerPoint slide

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TIFF original image Download: Fig 3. The ANDV-N protein enhances translation of the ANDV-like SmRNA in cells. HEK-293T cells were transfected with increasing amounts (50–500 ng) of a plasmid encoding for the recombinant ANDV His-N protein. Total transfected DNA was kept constant at 500 ng using pSP64 Poly(A) DNA. In vitro transcribed cap-N-RNA-3’UTR or cap-N-RNA-poly(A) RNAs, were transfected in cells together with a capped and polyadenylated mRNA encoding for RLuc (RLuc RNA). Six hours post-transfection (hpt), cells were lysed, and FLuc and RLuc activities were measured. (A) Expression of the ANDV His-N protein was confirmed by western blotting, using a monoclonal mouse anti-His antibody. GAPDH was used as a loading control. (B) The FLuc and RLuc activities were used to determine the relative translation efficiency (RTA), FLuc/RLuc, for each mRNA. Values shown are the mean (+/- SEM) of at least three independent experiments, each conducted in triplicate. Statistical analysis was performed by a two-way ANOVA with Sidak’s multiple comparisons test (P<0.05). https://doi.org/10.1371/journal.ppat.1009931.g003

The ANDV-N protein interacts with the eIF4G in cells Next, we wondered if, during infection, the ANDV-N and eIF4G proteins interacted in cells. For this, Huh-7 cells were infected with the ANDV CHI-7913 isolate (MOI of 1). Twenty-four hours post-infection, the expression of the ANDV-N and the endogenous eIF4G protein was evaluated by immunofluorescence (IF) (Fig 4A). The endogenous eIF4G protein showed a diffuse cytoplasmic distribution, while the distribution of the ANDV-N was also cytoplasmic but predominantly peri-nuclear (Fig 4A). We then performed an in situ proximity ligation assay (PLA) [40,41], using a rabbit anti-eIF4G and a mouse anti-ANDV-N as primary antibodies (Fig 4B). PLA recognizes target molecules in close proximity (<40 nm), and a positive signal is considered to reflect a protein-protein interaction [40,41]. ANDV-infected cells developed using only the secondary antibodies were used as negative controls (Fig 4A and 4B, panel’s upper right corner). The PLA signal (red spots) suggests that in ANDV-infected Huh-7 cells, the ANDV-N protein and the endogenous eIF4G interact (Fig 4B). PPT PowerPoint slide

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TIFF original image Download: Fig 4. The ANDV-N protein and eIF4G interact in ANDV infected Huh-7 cells. (A) Huh-7 cells were infected with ANDV (MOI 1), and 24 hours post-infection (hpi), cells were fixed (PFA 4%) and permeabilized (PBS-Triton). Expression of the ANDV-N and endogenous eIF4G proteins was confirmed by immunofluorescence (IF) using mouse monoclonal anti-ANDV-N or rabbit polyclonal anti-eIF4G as primary antibodies. As secondary antibodies, an Alexa 488 donkey anti-mouse or an Alexa 594 donkey anti-rabbit was used, respectively. As a negative control for the IF, the inset, in the upper right corner of the merge, shows ANDV infected cells where detection was conducted using only the secondary antibody. (B) ANDV-infected cells were incubated with primary antibodies as in (A), but PLA secondary antibodies were used following the manufacturer’s instructions. The inset, in the upper right corner of the merge, shows infected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of PLA. Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscope and processed by Image J. (C-D) HEK293T cells were cotransfected with the ANDV HA-N or HA-EV plasmids. Forty-eight hours later, cells were lysed, and immunoprecipitation (IP) assays were performed using protein A/G agarose coated with the corresponding antibody. The beads were washed and incubated with loading buffer at 95°C and the supernatant, which was used for western blotting. (C) Whole-cell lysate (WCL) of each sample and (D) IP fractions with the corresponding primary antibody highlighted on the right side of the panels. Western blotting was performed using mouse anti-HA, rabbit anti-eIF4G, and mouse anti-GAPDH. Horseradish Peroxidase (HRP)-conjugated Protein A/G was used to detect the primary antibodies. https://doi.org/10.1371/journal.ppat.1009931.g004 Next, the above experiment was repeated in a virus-free environment. For this, we carried out reciprocal co-immunoprecipitation (Co-IP) experiments in HEK 293T cells transfected with ANDV HA-N expression plasmid or the HA-EV control plasmid. Forty-eight hours later, cells were lysed (whole-cell lysate, WCL), and the expression of ANDV HA-N, and endogenous eIF4G was confirmed by western blotting using GAPDH as a loading control (Fig 4C). WCL were then subjected to immunoprecipitation using an anti-eIF4G or anti-HA antibody. Results show that the ANDV HA-N Co-IP with eIF4G when anti-eIF4G was used (Fig 4D). Likewise, eIF4G Co-IP with ANDV HA-N when the anti–HA antibody was used (Fig 4D). These results confirm that the ANDV-N and eIF4G interact in cells.

The human Mex 3A stimulates ANDV SmRNA translation Given that hMex3A-HA interacted with the (+)ANDV-SRNA in cells (Fig 6) and was identified as a SmRNA 3’UTR binding protein (S1 Data; DS3-DS4, DS1, and not in DS2), we next sought to evaluate if the protein participated in translation of the virus-like SmRNA in cells. For this, HEK 293T cells were transfected with a plasmid encoding the hMex3A-HA protein. Forty-eight hours later, cells were transfected with either the cap-N-RNA-3’UTR or the cap-N-RNA-poly(A) RNAs together with RLuc mRNA (used as a control for transfection efficiency). Six hours post mRNA transfection, the cells were lysed, and luciferase activities were determined and normalized to the RLuc control. The recombinant hMex3A-HA protein expression was confirmed by western blotting using an anti-HA antibody and detecting GAPDH as a loading control (Fig 7A). The FLuc/RLuc ratios obtained for the cap-N-RNA-3’UTR and the cap-N-RNA-poly(A) RNAs when used alone were set to 100%. In contrast, to what was reported for the cellular CDX2 mRNA [48], when overexpressed, the hMex3A-HA protein stimulated translation from the cap-N-RNA-3’UTR in a concentration-dependent manner (~120% increase at the maximum point) (Fig 7B). Interestingly, the expression of hMex3A-HA did not affect translation of the cap-N-RNA-poly(A) mRNA that lacks MREs (Fig 7B). Results showed that the impact of hMex3A-HA on the translation of the virus-like SmRNA was independent of the presence of viral proteins. Furthermore, results suggest that hMex3A-HA induced stimulation of the ANDV-like SmRNA was dependent on the presence of the ANDV SmRNA 3’UTR that harbors MREs (Fig 6A). This observation confirms that the 3’UTR of the SmRNA plays a role in translation, in this case, through the recruitment of at least one host protein. PPT PowerPoint slide

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TIFF original image Download: Fig 7. The recombinant HA-hMex 3A protein enhances translation of ANDV-like SmRNA in cells. HEK-293T cells were transfected with increasing amounts (50–500 ng) of a plasmid encoding for the recombinant hMex3A-HA protein. Total transfected DNA was kept constant at 500 ng using pSP64 Poly(A) DNA. The cap-N-RNA-3’UTR or cap-N-RNA-poly(A) RNAs were transfected in cells together with a capped and polyadenylated mRNA encoding for RLuc. Six hpt, cells were lysed, and FLuc and RLuc activities were measured. (A) Expression of the hMex3A-HA protein was confirmed by western blotting, using a monoclonal mouse anti-HA antibody. GAPDH was used as a loading control. (B) The FLuc and RLuc activities were used to determine the RTA for each mRNA. Values shown are the mean (+/- SEM) of at least three independent experiments, each conducted in triplicate. Statistical analysis was performed by a two-way ANOVA with Sidak’s multiple comparisons test (P<0.05). https://doi.org/10.1371/journal.ppat.1009931.g007

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009931

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