The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

The NS2B-PP1α-eIF2α axis: Inhibiting stress granule formation and Boosting Zika virus replication.

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.

Vimentin inhibits α-tubulin acetylation via enhancing α-TAT1 degradation to suppress the replication of human parainfluenza virus type 3.

We previously found that, among human parainfluenza virus type 3 (HPIV3) proteins, the interaction of nucleoprotein (N) and phosphoprotein (P) provides the minimal requirement for the formation of cytoplasmic inclusion bodies (IBs), which are sites of RNA synthesis, and that acetylated α-tubulin enhances IB fusion and viral replication. In this study, using immunoprecipitation and mass spectrometry assays, we determined that vimentin (VIM) specifically interacted with the N-P complex of HPIV3, and that the head domain of VIM was responsible for this interaction, contributing to the inhibition of IB fusion and viral replication. Furthermore, we found that VIM promoted the degradation of α-tubulin acetyltransferase 1 (α-TAT1), through its head region, thereby inhibiting the acetylation of α-tubulin, IB fusion, and viral replication. In addition, we identified a 20-amino-acid peptide derived from the head region of VIM that participated in the interaction with the N-P complex and inhibited viral replication. Our findings suggest that VIM inhibits the formation of HPIV3 IBs by downregulating α-tubulin acetylation via enhancing the degradation of α-TAT1. Our work sheds light on a new mechanism by which VIM suppresses HPIV3 replication.

SLC35B2 Acts in a Dual Role in the Host Sulfation Required for EV71 Infection.

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. IMPORTANCE As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.

P300-mediated NEDD4 acetylation drives ebolavirus VP40 egress by enhancing NEDD4 ligase activity.

The final stage of Ebola virus (EBOV) replication is budding from host cells, where the matrix protein VP40 is essential for driving this process. Many post-translational modifications such as ubiquitination are involved in VP40 egress, but acetylation has not been studied yet. Here, we characterize NEDD4 is acetylated at a conserved Lys667 mediated by the acetyltransferase P300 which drives VP40 egress process. Importantly, P300-mediated NEDD4 acetylation promotes NEDD4-VP40 interaction which enhances NEDD4 E3 ligase activity and is essential for the activation of VP40 ubiquitination and subsequent egress. Finally, we find that Zaire ebolavirus production is dramatically reduced in P300 knockout cell lines, suggesting that P300-mediated NEDD4 acetylation may have a physiological effect on Ebola virus life cycle. Thus, our study identifies an acetylation-dependent regulatory mechanism that governs VP40 ubiquitination and provides insights into how acetylation controls EBOV VP40 egress.

[Characterization of Cell Subsets Associated With Prognosis of Osteosarcoma Based on Single-Cell Sequencing Data].

Objective To explore the cell subsets and characteristics related to the prognosis of osteosarcoma by analyzing the cellular composition of tumor tissue samples from different osteosarcoma patients.Methods The single-cell sequencing data and bulk sequencing data of different osteosarcoma patients were downloaded.We extracted the information of cell samples for dimensionality reduction,annotation,and cell function analysis,so as to identify the cell subsets and clarify the cell characteristics related to the prognosis of osteosarcoma.The development trajectory of macrophages with prognostic significance was analyzed,and the prognostic model of osteosarcoma was established based on the differentially expressed genes of macrophage differentiation.Results The cellular composition presented heterogeneity in the patients with osteosarcoma.The infiltration of mononuclear phagocytes in osteosarcoma had prognostic significance(P=0.003).Four macrophage subsets were associated with prognosis,and their signature transcription factors included RUNX3(+),ETS1(+),HOXD11(+),ZNF281(+),and PRRX1(+).Prog_Macro2 and Prog_Macro4 were located at the end of the developmental trajectory,and the prognostic ability of macrophage subsets increased with the progression of osteosarcoma.The prognostic model established based on the differentially expressed genes involved in macrophage differentiation can distinguish the survival rate of osteosarcoma patients with different risks(P<0.001).Conclusion Macrophage subsets are closely related to the prognosis of osteosarcoma and can be used as the key target cells for the immunotherapy of osteosarcoma.

Early prediction of sepsis using double fusion of deep features and handcrafted features.

Sepsis is a life-threatening medical condition that is characterized by the dysregulated immune system response to infections, having both high morbidity and mortality rates. Early prediction of sepsis is critical to the decrease of mortality. This paper presents a novel early warning model called Double Fusion Sepsis Predictor (DFSP) for sepsis onset. DFSP is a double fusion framework that combines the benefits of early and late fusion strategies. First, a hybrid deep learning model that combines both the convolutional and recurrent neural networks to extract deep features is proposed. Second, deep features and handcrafted features, such as clinical scores, are concatenated to build the joint feature representation (early fusion). Third, several tree-based models based on joint feature representation are developed to generate the risk scores of sepsis onset that are combined with an End-to-End neural network for final sepsis detection (late fusion). To evaluate DFSP, a retrospective study was conducted, which included patients admitted to the ICUs of a hospital in Shanghai China. The results demonstrate that the DFSP outperforms state-of-the-art approaches in early sepsis prediction.

Enterovirus 71 2A Protease Inhibits P-Body Formation To Promote Viral RNA Synthesis.

Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here, we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proven by the result that infection with EV71-2AC110S, a 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we show that DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and nascent RNA capture assay. Altogether, our data first demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulation of gene expression and mRNA degradation. The P-body is the structure that viruses manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection, and its activity is essential. When the assembly of P-bodies was blocked by 2A protease, DDX6 and 4E-T, which were required for P-body formation, bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs.

Viral invasion triggers the activation of the host antiviral response. Besides the innate immune response, stress granules (SGs) also act as an additional defense response to combat viral replication. However, many viruses have evolved various strategies to suppress SG formation to facilitate their own replication. Here, we show that viral mRNAs derived from human parainfluenza virus type 3 (HPIV3) infection induce SG formation in an eIF2α phosphorylation- and PKR-dependent manner in which viral mRNAs are sequestered and viral replication is inhibited independent of the interferon signaling pathway. Furthermore, we found that inclusion body (IB) formation by the interaction of the nucleoprotein (N) and phosphoprotein (P) of HPIV3 correlated with SG suppression. In addition, co-expression of P with NL478A (a point mutant of N, which is unable to form IBs with P) or with NΔN10 (lacking N-terminal 10 amino acids of N, which could form IBs with P but was unable to synthesize or shield viral RNAs) failed to inhibit SG formation, suggesting that inhibition of SG formation also correlates with the capacity of IBs to synthesize and shield viral RNAs. Therefore, we provide a model whereby viral IBs escape the antiviral effect of SGs by concealing their own newly synthesized viral RNAs and offer new insights into the emerging role of IBs in viral replication.

Picornavirus 2A protease regulates stress granule formation to facilitate viral translation.

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation.

Femtometer-resolved simultaneous measurement of multiple laser wavelengths in a speckle wavemeter.

Many areas of optical science require an accurate measurement of optical spectra. Devices based on laser speckle promise compact wavelength measurement, with attometer-level sensitivity demonstrated for single wavelength laser fields. The measurement of multimode spectra using this approach would be attractive, yet this is currently limited to picometer resolution. Here, we present a method to improve the resolution and precision of speckle-based multi-wavelength measurements. We measure multiple wavelengths simultaneously, in a device comprising a single 1-m-long step-index multimode fiber and a fast camera. Independent wavelengths separated by as little as 1 fm are retrieved with 0.2 fm precision using principal component analysis. The method offers a viable way to measure sparse spectra containing multiple individual lines and may find application in the tracking of multiple lasers in fields such as quantum technologies and optical telecommunications.

Label-free optical hemogram of granulocytes enhanced by artificial neural networks.

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.

Overcoming the speckle correlation limit to achieve a fiber wavemeter with attometer resolution.

The measurement of the wavelength of light using speckle is a promising tool for the realization of compact and precise wavemeters and spectrometers. However, the resolution of these devices is limited by strong correlations between the speckle patterns produced by closely spaced wavelengths. Here, we show how principal component analysis of speckle images provides a route to overcome this limit. Using this, we demonstrate a compact wavemeter that measures attometer-scale wavelength changes of a stabilized diode laser, eight orders of magnitude below the speckle correlation limit.

Optimal compressive multiphoton imaging at depth using single-pixel detection.

Compressive sensing can overcome the Nyquist criterion and record images with a fraction of the usual number of measurements required. However, conventional measurement bases are susceptible to diffraction and scattering, prevalent in high-resolution microscopy. In this Letter, we explore the random Morlet basis as an optimal set for compressive multiphoton imaging, based on its ability to minimize the space-frequency uncertainty. We implement this approach for wide-field multiphoton microscopy with single-pixel detection, which allows imaging through turbid media without correction. The Morlet basis promises a route for rapid acquisition with lower photodamage.

Inclusion Body Fusion of Human Parainfluenza Virus Type 3 Regulated by Acetylated α-Tubulin Enhances Viral Replication.

Viral inclusion bodies (IBs), or replication factories, are unique structures generated by viral proteins together with some cellular proteins as a platform for efficient viral replication, but little is known about the mechanism underlying IB formation and fusion. Our previous study demonstrated that the interaction between the nucleoprotein (N) and phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3), an enveloped virus with great medical impact, can form IBs. In this study, we found that small IBs can fuse with each other to form large IBs that enhance viral replication. Furthermore, we found that acetylated α-tubulin interacts with the N-P complex and colocalizes with IBs of HPIV3 but does not interact with the N-P complex of human respiratory syncytial virus or vesicular stomatitis virus and does not colocalize with IBs of human respiratory syncytial virus. Most importantly, enhancement of α-tubulin acetylation using the pharmacological inhibitor trichostatin A (TSA), RNA interference (RNAi) knockdown of the deacetylase enzymes histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), or expression of α-tubulin acetyltransferase 1 (α-TAT1) resulted in the fusion of small IBs into large IBs and effective viral replication. In contrast, suppression of acetylation of α-tubulin by overexpressing HDAC6 and SIRT2 profoundly inhibited the fusion of small IBs and viral replication. Our findings offer previously unidentified mechanistic insights into the regulation of viral IB fusion by acetylated α-tubulin, which is critical for viral replication. IMPORTANCE: Inclusion bodies (IBs) are unique structures generated by viral proteins and some cellular proteins as a platform for efficient viral replication. Human parainfluenza virus type 3 (HPIV3) is a nonsegmented single-stranded RNA virus that mainly causes lower respiratory tract disease in infants and young children. However, no vaccines or antiviral drugs for HPIV3 are available. Therefore, understanding virus-host interactions and developing new antiviral strategies are increasingly important. Acetylation on lysine (K) 40 of α-tubulin is an evolutionarily conserved modification and plays an important role in many cellular processes, but its role in viral IB dynamics has not been fully explored. To our knowledge, our findings are the first to show that acetylated α-tubulin enhances viral replication by regulating HPIV3 IB fusion.

Optical trapping with planar silicon metalenses.

Contactless manipulation of micron-scale objects in a microfluidic environment is a key ingredient for a range of applications in the biosciences, including sorting, guiding, and analysis of cells and bacteria. Optical forces are powerful for this purpose but, typically, require bulky focusing elements to achieve the appropriate optical field gradients. To this end, realizing the focusing optics in a planar format would be very attractive and conducive to the integration of such microscale devices, either individually or as arrays. Here we report on, to the best of our knowledge, the first experimental demonstration of optical trapping using planar silicon metalenses illuminated with a collimated laser beam. The structures consist of high-contrast gratings with a locally varying period and duty cycle. They are designed to mimic parabolic reflectors with a numerical aperture of 0.56 at a vacuum wavelength of 1064 nm. We achieve both two- and three-dimensional trapping in water, with the latter realized by omitting the central Fresnel zones. This Letter highlights the versatility of such lithographically defined metastructures for exerting optical forces without the need for traditional optical elements.

Raman imaging through a single multimode fibre.

Vibrational spectroscopy is a widespread, powerful method of recording the molecular spectra of constituent molecules within a sample in a label-free manner. As an example, Raman spectroscopy has major applications in materials science, biomedical analysis and clinical studies. The need to access deep tissues and organs in vivo has triggered major advances in fibre Raman probes that are compatible with endoscopic settings. However, imaging in confined geometries still remains out of reach for the current state of art fibre Raman systems without compromising the compactness and flexibility. Here we demonstrate Raman spectroscopic imaging via complex correction in single multimode fibre without using any additional optics and filters in the probe design. Our approach retains the information content typical to traditional fibre bundle imaging, yet within an ultra-thin footprint of diameter 125 µm which is the thinnest Raman imaging probe realised to date. We are able to acquire Raman images, including for bacteria samples, with fields of view exceeding 200 µm in diameter.

Casein kinase II controls TBK1/IRF3 activation in IFN response against viral infection.

By sensing viral nucleic acids, host innate receptors elicit signaling pathways converging on TBK1-IFN regulatory factor (IRF)3 axis in mediating IFN-αß induction and defense mechanisms. In contrast, viruses have evolved with diverse immune evasion/interference mechanisms to undermine innate receptor signaling and IFN response. In this regard, approaches enabling host to overcome such immune evasion/interference mechanisms are urgently needed to combat infections by epidemic/pandemic viruses. In this study, we report that protein kinase CK2 serves as a key component controlling TBK1 and IRF3 activation in IFN-inducing TLR, RIG-I-like receptors, and cGAS/STING signaling pathways. Accordingly, knocking down of CK2 expression or genetic ablation of its kinase activity resulted in elevated IFN-αß response in response to infection by DNA and RNA viruses. Moreover, PP2A was identified as one of the intermediate phosphatases responsible for CK2-regulated IFN response, suggesting that CK2 may regulate TBK1 and IRF3 activation indirectly. Importantly, blockade of CK2 activity by small molecule inhibitor was able to activate TBK1, whereby eliciting effective host defense mechanisms against hepatitis C virus infection. Taken together, our results identify CK2 as a novel regulator of TBK1 and IRF3 and suggest that targeting CK2 by small molecular inhibitor may be a viable approach to prevent and treat viral infections.

Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly.

UNLABELLED: Human parainfluenza virus type 3 (HPIV3) belongs to the Paramyxoviridae family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (ML305A) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the ML305A mutation (rHPIV3-ML305A) could be successfully recovered. In the present study, we found that the titer of rHPIV3-ML305A was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and ML305A-VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into ML305A-VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into ML305A-VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles. IMPORTANCE: Human parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the Paramyxoviridae family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.

A leucine residue in the C terminus of human parainfluenza virus type 3 matrix protein is essential for efficient virus-like particle and virion release.

UNLABELLED: Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the ML302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M(L302A) mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. IMPORTANCE: Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the M(L302A) mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.