Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

Autophagic membranes participate in hepatitis B virus nucleocapsid assembly, precore and core protein trafficking, and viral release.

Hepatitis B virus (HBV) DNA replication takes place inside the viral core particle and is dependent on autophagy. Here we show that HBV core particles are associated with autophagosomes and phagophores in cells that productively replicate HBV. These autophagic membrane-associated core particles contain almost entirely the hypophosphorylated core protein and are DNA replication competent. As the hyperphosphorylated core protein can be localized to phagophores and the dephosphorylation of the core protein is associated with the packaging of viral pregenomic RNA (pgRNA), these results are in support of the model that phagophores can serve as the sites for the packaging of pgRNA. In contrast, in cells that replicate HBV, the precore protein derivatives, which are related to the core protein, are associated with autophagosomes but not with phagophores via a pathway that is independent of its signal peptide. Interestingly, when the core protein is expressed by itself, it is associated with phagophores but not with autophagosomes. These observations indicate that autophagic membranes are differentially involved in the trafficking of precore and core proteins. HBV induces the fusion of autophagosomes and multivesicular bodies and the silencing of Rab11, a regulator of this fusion, is associated with the reduction of release of mature HBV particles. Our studies thus indicate that autophagic membranes participate in the assembly of HBV nucleocapsids, the trafficking of HBV precore and core proteins, and likely also the egress of HBV particles.

Human Cytomegalovirus Egress: Overcoming Barriers and Co-Opting Cellular Functions.

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.

Nucleocapsid Structure of Negative Strand RNA Virus.

Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein-RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein-RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication.

Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly.

Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor.IMPORTANCE The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro, but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.

Vesicular stomatitis virus nucleocapsids diffuse through cytoplasm by hopping from trap to trap in random directions.

Within 2-6 hours after infection by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of infected cells. In that time, viral ribonucleoprotein (RNP) particles (nucleocapsids) travel from their initial sites of synthesis near the nucleus to the edge of the cell, a distance of 5-10 µm. The hydrodynamic radius of RNP particles (86 nm) precludes simple diffusion through the mesh of cytoskeletal fibers. To reveal the relative importance of different transport mechanisms, movement of GFP-labeled RNP particles in live A549 cells was recorded within 3 to 4 h postinfection at 100 frames/s by fluorescence video microscopy. Analysis of more than 200 RNP particle tracks by Bayesian pattern recognition software found that 3% of particles showed rapid, directional motion at about 1 µm/s, as previously reported. 97% of the RNP particles jiggled within a small, approximately circular area with Gaussian width σ = 0.06 µm. Motion within such "traps" was not directional. Particles stayed in traps for approximately 1 s, then hopped to adjacent traps whose centers were displaced by approximately 0.17 µm. Because hopping occurred much more frequently than directional motion, overall transport of RNP particles was dominated by hopping over the time interval of these experiments.

Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35.

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain.IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

Disrupting the association of Autographa californica multiple nucleopolyhedrovirus Ac93 with cellular ESCRT-III/Vps4 hinders nuclear egress of nucleocapsids and intranuclear microvesicles formation.

The endosomal sorting complex required for transport (ESCRT) pathway is required for efficient egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this study, we found that Ac93, a baculovirus core protein, contains a conserved MIM1-like motif. Alanine substitutions for six leucine residues in MIM1-like motif revealed that L142, L145, L146, and L149 are required for association of Ac93 with the MIT domain of Vps4. Mutations of these residues also blocked self-association and the association of Ac93 with ESCRT-III proteins or other viral core proteins Ac76 and Ac103, and resulted in a substantial reduction of infectious virus production, less efficient nuclear egress of progeny nucleocapsids, and the defect of intranuclear microvesicles formation. Combined with the localization of the association of Ac93 with ESCRT-III/Vps4 and other viral proteins at the nuclear membrane, we propose that the coordinated action of these viral proteins and ESCRT-III/Vps4 may be involved in remodeling the nuclear membrane.

Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome.

Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

AcMNPV PKIP is associated with nucleocapsid of budded virions and involved in nucleocapsid assembly.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf24 (pkip) is a unique Alphabaculovirus gene. A previous study showed that a temperature-sensitive mutant of AcMNPV with a mutation in pkip displayed severe defects in progeny budded virion (BV) production and very late gene transcription, however, the underlying mechanism has not been determined. To investigate the function of pkip in the baculovirus replication cycle, we constructed a pkip-knockout AcMNPV bacmid in this study. Our results showed that deletion of pkip led to significant reduction of BV production, while the synthesis of viral DNA and the transcription of early and late genes were not affected. Further examination by transmission electron microscopy analysis showed that deletion of pkip resulted in the formation of massive electron-lucent tubular structures in the nucleus of the infected cells, along with some normal electron-dense nucleocapsids. The pkip-encoded protein PKIP could be detected at late phase during infection and was distributed in both the cytoplasm and nuclei of viruses-infected cells, with a ring pattern near the inner nuclear membrane and punctate distribution in the virogenic stroma area. Biochemical fractionation of virions into nucleocapsid and envelop components showed that PKIP was associated with the nucleocapsid fraction of BV. Taken together, our results indicated that PKIP is associated with nucleocapsids of BV and involved in nucleocapsid assembly, which contributes to the optimal production of BV.

Disruption of Autographa Californica Multiple Nucleopolyhedrovirus ac111 Results in Reduced per os Infectivity in a Host-Dependent Manner.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac111 gene is highly conserved in lepidopteran-specific baculoviruses, and its function in the AcMNPV life cycle is still unknown. To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner.

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions.

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.

Bombyx mori nucleopolyhedrovirus orf133 and orf134 are involved in the embedding of occlusion-derived viruses into polyhedra.

Bombyx mori nucleopolyhedrovirus (BmNPV) orf133 (bm133) and orf134 (bm134), the orthologues of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac4 and ac5, are two adjacent genes with opposite transcriptional orientations and are highly conserved in all sequenced group I nucleopolyhedroviruses (NPVs). A double bm133-bm134 knockout bacmid was generated to enable the functional study of each gene independently or together. Compared with wild-type and double-repair viruses, deletion of both bm133 and bm134 did not affect budded virus (BV) production or viral DNA replication in transfected BmN cells. Electron microscopy revealed that the double knockout did not affect nucleocapsid assembly, virus-induced intranuclear microvesicle formation or occlusion-derived virus (ODV) production, but the number of virions embedded in the polyhedra decreased significantly. Further investigations showed that disruption of either gene was unable to recover the defect of ODV occlusion, suggesting that Bm133 and Bm134 are indispensable to the embedding of ODVs into polyhedra. Confocal microscopy analysis showed that Bm133 and Bm134 distributed throughout the whole cell during viral infection and Bm134 concentrated on the mature polyhedra in lysed cells. These results suggest that although Bm133 and Bm134 are not essential for BV or ODV development, they play vital roles in polyhedra morphogenesis.

The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly.

UNLABELLED: Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly. IMPORTANCE: Baculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

UNLABELLED: Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE: Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.

Influenza virus adaptation PB2-627K modulates nucleocapsid inhibition by the pathogen sensor RIG-I.

The cytoplasmic RNA helicase RIG-I mediates innate sensing of RNA viruses. The genomes of influenza A virus (FLUAV) are encapsidated by the nucleoprotein and associated with RNA polymerase, posing potential barriers to RIG-I sensing. We show that RIG-I recognizes the 5'-triphosphorylated dsRNA on FLUAV nucleocapsids but that polymorphisms at position 627 of the viral polymerase subunit PB2 modulate RIG-I sensing. Compared to mammalian-adapted PB2-627K, avian FLUAV nucleocapsids possessing PB2-627E are prone to increased RIG-I recognition, and RIG-I-deficiency partially restores PB2-627E virus infection of mammalian cells. Heightened RIG-I sensing of PB2-627E nucleocapsids correlates with previously established lower affinity of 627E-containing PB2 for nucleoprotein and is increased by further nucleocapsid instability. The effect of RIG-I on PB2-627E nucleocapsids is independent of antiviral signaling, suggesting that RIG-I-nucleocapsid binding alone can inhibit infection. These results indicate that RIG-I is a direct avian FLUAV restriction factor and highlight nucleocapsid disruption as an antiviral strategy.

Differential contributions of tacaribe arenavirus nucleoprotein N-terminal and C-terminal residues to nucleocapsid functional activity.

UNLABELLED: The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE: The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding.

Viral and host control of cytomegalovirus maturation.

Maturation in herpesviruses initiates in the nucleus of the infected cell, with encapsidation of viral DNA to form nucleocapsids, and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize current knowledge of and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses, with an emphasis on viral and host factors that regulate this process.

Evaluation of methods measuring the capsid integrity and/or functions of noroviruses by heat inactivation.

Attempts were made to evaluate methods measuring the capsid integrity and/or functions of noroviruses (NoVs) following heat treatment. Intact viruses (Murine Norovirus-1 [MNV-1] and human NoV GII.4), virus like particles (VLPs) and P particles (expressed in vitro from the protruding domain of the viral capsid) of NoVs were used in this study. Following heat treatment, no significant difference of viral titer of MNV-1 versus NoV GII.4 was observed by RNase One RT-PCR or cell-binding RT-PCR, although cell-binding RT-PCR (to measure the capsid functions) revealed higher reductions than RNase One RT-PCR (to measure the capsid integrity). These results indicate that the function assay for receptor binding is more sensitive than the capsid integrity assay to measure the protected viral RNA. MNV-1 could be used as a surrogate for human NoVs by heat inactivation from the perspective of capsid integrity and/or functions. The heat resistance varied among different GI and GII NoV strains when their P particles were studied.

Knockdown of Ebola virus VP24 impairs viral nucleocapsid assembly and prevents virus replication.

The structural protein VP24 of Ebola virus (EBOV) is a determinant of virulence in rodent models and possesses an interferon antagonist function. In this study, we investigate the role of VP24 in EBOV replication using RNA interference by small interfering RNA to knock down the expression of this protein in virus-infected cells. We reveal that VP24 is required for assembly of viral nucleocapsids and that silencing of VP24 expression prevents the release of EBOV.