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Nature Communications Sep 2023Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template...
Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation.
Topics: Child; Aged; Humans; Respiratory Syncytial Virus, Human; Cryoelectron Microscopy; Nucleocapsid; RNA, Viral; Nucleoproteins
PubMed: 37714861
DOI: 10.1038/s41467-023-41439-8 -
Journal of Virology Sep 2022Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and...
Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.
Topics: Biological Transport; Ebolavirus; Hemorrhagic Fever, Ebola; Humans; Nucleocapsid; Phosphorylation; Transcription Factors; Viral Proteins; Virion; Virus Assembly
PubMed: 35993739
DOI: 10.1128/jvi.01083-22 -
Genes Feb 2022The genus , consisting of four main subgenera (, , and ), encompasses all clinically significant coronaviruses (CoVs), including SARS, MERS, and the SARS-CoV-2 virus...
The genus , consisting of four main subgenera (, , and ), encompasses all clinically significant coronaviruses (CoVs), including SARS, MERS, and the SARS-CoV-2 virus responsible for current COVID-19 pandemic. Very few molecular characteristics are known that are specific for the genus or its different subgenera. In this study, our analyses of the sequences of four essential proteins of CoVs, viz., spike, nucleocapsid, envelope, and RNA-dependent RNA polymerase (RdRp), identified ten novel molecular signatures consisting of conserved signature indels (CSIs) in these proteins which are specific for the genus or its subgenera. Of these CSIs, two 14-aa-conserved deletions found within the heptad repeat motifs 1 and 2 of the spike protein are specific for all betacoronaviruses, except for their shared presence in the highly infectious avian coronavirus. Six additional CSIs present in the nucleocapsid protein and one CSI in the RdRp protein are distinctive characteristics of either the , or subgenera. In addition, a 4-aa insert is present in the spike protein, which is uniquely shared by all viruses from the subgenera , and , but absent in and all other genera of CoVs. This molecular signature provides evidence that viruses from the three subgenera sharing this CSI are more closely related to each other, and they evolved after the divergence of embecoviruses and other CoVs. As all CSIs specific for different groups of CoVs are flanked by conserved regions, their sequences provide novel means for identifying the above groups of CoVs and for developing novel diagnostic tests. Furthermore, our analyses of the structures of the spike and nucleocapsid proteins show that all identified CSIs are localized in the surface-exposed loops of these protein. It is postulated that these surface loops, through their interactions with other cellular proteins/ligands, play important roles in the biology/pathology of these viruses.
Topics: COVID-19; Humans; Nucleocapsid; Pandemics; Phylogeny; SARS-CoV-2
PubMed: 35327976
DOI: 10.3390/genes13030423 -
Viruses Dec 2021The assembly of human cytomegalovirus (HCMV) and other herpesviruses includes both nuclear and cytoplasmic phases. During the prolonged replication cycle of HCMV, the... (Review)
Review
The assembly of human cytomegalovirus (HCMV) and other herpesviruses includes both nuclear and cytoplasmic phases. During the prolonged replication cycle of HCMV, the cell undergoes remarkable changes in cellular architecture that include marked increases in nuclear size and structure as well as the reorganization of membranes in cytoplasm. Similarly, significant changes occur in cellular metabolism, protein trafficking, and cellular homeostatic functions. These cellular modifications are considered integral in the efficient assembly of infectious progeny in productively infected cells. Nuclear egress of HCMV nucleocapsids is thought to follow a pathway similar to that proposed for other members of the herpesvirus family. During this process, viral nucleocapsids must overcome structural barriers in the nucleus that limit transit and, ultimately, their delivery to the cytoplasm for final assembly of progeny virions. HCMV, similar to other herpesviruses, encodes viral functions that co-opt cellular functions to overcome these barriers and to bridge the bilaminar nuclear membrane. In this brief review, we will highlight some of the mechanisms that define our current understanding of HCMV egress, relying heavily on the current understanding of egress of the more well-studied α-herpesviruses, HSV-1 and PRV.
Topics: Capsid; Cell Nucleus; Cytomegalovirus; Cytoplasm; DNA Replication; DNA, Viral; Humans; Nuclear Envelope; Nucleocapsid; Viral Genome Packaging; Virus Release; Virus Replication
PubMed: 35062219
DOI: 10.3390/v14010015 -
The Journal of Biological Chemistry Mar 2022In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For... (Review)
Review
In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.
Topics: COVID-19; Consensus; Humans; Nucleocapsid; Nucleocapsid Proteins; RNA, Viral; SARS-CoV-2; Viral Proteins
PubMed: 35131265
DOI: 10.1016/j.jbc.2022.101677 -
Viruses Dec 2021All paramyxoviruses, which include the mumps virus, measles virus, Nipah virus, Newcastle disease virus, and Sendai virus, have non-segmented single-stranded... (Review)
Review
All paramyxoviruses, which include the mumps virus, measles virus, Nipah virus, Newcastle disease virus, and Sendai virus, have non-segmented single-stranded negative-sense RNA genomes. These RNA genomes are enwrapped throughout the viral life cycle by nucleoproteins, forming helical nucleocapsids. In addition to these helical structures, recombinant paramyxovirus nucleocapsids may occur in other assembly forms such as rings, clam-shaped structures, and double-headed nucleocapsids; the latter two are composed of two single-stranded helices packed in a back-to-back pattern. In all of these assemblies, the neighboring nucleoprotein protomers adopt the same domain-swapping mode via the N-terminal arm, C-terminal arm, and recently disclosed N-hole. An intrinsically disordered region in the C-terminal domain of the nucleoproteins, called the N-tail, plays an unexpected role in regulating the transition among the different assembly forms that occurs with other viral proteins, especially phosphoprotein. These structures, together with the helical nucleocapsids, significantly enrich the structural diversity of the paramyxovirus nucleocapsids and help explain the functions of these diverse assemblies, including RNA genome protection, transcription, and replication, as well as encapsulation.
Topics: Models, Molecular; Nucleocapsid; Nucleocapsid Proteins; Paramyxovirinae; Protein Conformation; Protein Domains; Protein Structure, Quaternary; Protein Subunits
PubMed: 34960748
DOI: 10.3390/v13122479 -
Viruses Feb 2023Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to... (Review)
Review
Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication. Structural determination of RNPs can provide crucial information on the molecular mechanisms of these processes, paving the way for the development of new, more effective strategies to control and prevent the spread of ssRNAv diseases. In this scenario, cryogenic electron microscopy (cryoEM), relying on the technical and methodological revolution it has undergone in recent years, can provide invaluable help in elucidating how these macromolecular complexes are organized, packaged within the virion, or the functional implications of these structures. In this review, we summarize some of the most prominent achievements by cryoEM in the study of RNP and nucleocapsid structures in lipid-enveloped ssRNAv.
Topics: Humans; RNA, Viral; Cryoelectron Microscopy; Ribonucleoproteins; Viral Proteins; Nucleocapsid; Influenza A virus
PubMed: 36992363
DOI: 10.3390/v15030653 -
Communications Biology Apr 2021Paramyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are...
Paramyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.
Topics: Cryoelectron Microscopy; Nucleocapsid; Nucleoproteins; Sendai virus; Viral Proteins
PubMed: 33888861
DOI: 10.1038/s42003-021-02027-y -
Journal of Virology May 2022Herpesviruses assemble new viral particles in the nucleus. These nucleocapsids bud through the inner nuclear membrane to produce enveloped viral particles in the...
Herpesviruses assemble new viral particles in the nucleus. These nucleocapsids bud through the inner nuclear membrane to produce enveloped viral particles in the perinuclear space before fusing with the outer nuclear membrane to reach the cytoplasm. This unusual route is necessary since viral capsids are too large to pass through nuclear pores. However, the transient perinuclear nucleocapsids (250 nm in diameter) are also larger than the width of the perinuclear space (30 to 50 nm). Interestingly, linker of the nucleoskeleton and cytoskeleton (LINC) components SUN and KASH connect the inner and outer nuclear membranes and regulate their spacing. Previous work by others on the related pseudorabies virus and human cytomegalovirus showed that they functionally interact with SUN proteins. To clarify the role of SUN proteins, we explored their impact on herpes simplex virus 1 (HSV-1), another herpesvirus. Using dominant negative SUN mutants and RNA interference, we show that HSV-1 propagation is dependent on the LINC complex. In contrast to pseudorabies virus, SUN2 disruption by either approach led to increased HSV-1 extracellular viral yields. This SUN2 dependency may be linked to its greater impact on perinuclear spacing in infected cells compared to SUN1. Finally, the virus itself seems to modulate perinuclear spacing. The large size of herpesviruses prevents them from travelling across the nuclear pores, and they instead egress across the two nuclear membranes, generating short-lived enveloped perinuclear virions. This poses a challenge as the perinuclear space is smaller than the virions. This implies the separation (unzipping) of the two nuclear membranes to accommodate the viral particles. The LINC complex bridges the two nuclear membranes and is an important regulator of perinuclear spacing. Work by others hint at its functional implication during pseudorabies virus and cytomegalovirus propagation. The present study probes the importance for HSV-1 of the SUN proteins, the LINC components found in the inner nuclear membrane. Using dominant negative constructs and RNA interference (RNAi), the data reveal that SUN2 exhibits antiviral propriety toward HSV-1, as disrupting the protein leads to increased viral yields. This is in contrast with that reported for pseudorabies and suggests that differences among herpesviruses may, once again, prevail.
Topics: Animals; Cell Nucleus; Herpesvirus 1, Human; Herpesvirus 1, Suid; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nuclear Envelope; Nucleocapsid; Virion
PubMed: 35435724
DOI: 10.1128/jvi.00453-22 -
Protein & Cell May 2019Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human... (Review)
Review
Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human pathogens and cause many diseases. As large enveloped DNA viruses, herpesviruses undergo several complex steps to complete their life cycles and produce infectious progenies. Firstly, herpesvirus assembly initiates in the nucleus, producing nucleocapsids that are too large to cross through the nuclear pores. Nascent nucleocapsids instead bud at the inner nuclear membrane to form primary enveloped virions in the perinuclear space followed by fusion of the primary envelopes with the outer nuclear membrane, to translocate the nucleocapsids into the cytoplasm. Secondly, nucleocapsids obtain a series of tegument proteins in the cytoplasm and bud into vesicles derived from host organelles to acquire viral envelopes. The vesicles are then transported to and fuse with the plasma membrane to release the mature virions to the extracellular space. Therefore, at least two budding and fusion events take place at cellular membrane structures during herpesviruses assembly and egress, which induce membrane deformations. In this review, we describe and discuss how herpesviruses exploit and remodel host membrane structures to assemble and escape from the host cell.
Topics: Animals; Herpesviridae; Herpesviridae Infections; Host Microbial Interactions; Humans; Nuclear Envelope; Nucleocapsid; Viral Proteins; Virus Assembly; Virus Release
PubMed: 30242641
DOI: 10.1007/s13238-018-0577-9