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Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5′ end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m⁶A)–modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m⁶A modification of the 5′ ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m⁶A sitemutant in the HBV 5′ epsilon, we present evidence that m⁶A methylation of 5′ epsilon is necessary for its encapsidation. The m⁶A modification of 5′ epsilon increased the efficiency of viral RNA packaging, whereas the m⁶A of 3′ epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m⁶A modification at 5′ epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5′ epsilon m⁶A site–mutated pgRNA failed to interact. HBV polymerase interaction with 5′ epsilon was independent of m⁶A modification of 5′ epsilon. This study highlights yet another pivotal role of m⁶A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA–protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m⁶A modification.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus that causes severe hemorrhagic fever, leukopenia, thrombocytopenia, and multi-organ failure, with a case fatality rate of up to 30%. No licensed vaccines or specific antiviral therapies are currently available. The viral nucleocapsid protein (NP) is essential for viral transcription and replication, forming a ribonucleoprotein complex (RNP) by encapsidating viral genomic RNA. However, the structural basis of RNA recognition and encapsidation by SFTSV NP remains poorly understood. In this study, we determined a cryo-electron microscopy structure of the SFTSV NP-RNA complex. Structural comparisons and evolutionary conservation analysis of NPs across the family Phenuiviridae uncovered a conserved RNA-binding mode among phenuiviruses, suggesting a shared RNA encapsidation mechanism among related viruses. Our findings provide critical structural insights into SFTSV RNA encapsidation and will aid future efforts to develop antivirals against SFTSV and related pathogenic viruses.

Hantaviruses infect a wide range of hosts including insectivores and rodents and can also cause zoonotic infections in humans, which can lead to severe disease with possible fatal outcomes. Hantavirus outbreaks are usually linked to the population dynamics of the host animals and their habitats being in close proximity to humans, which is becoming increasingly important in a globalized world. Currently there is neither an approved vaccine nor a specific and effective antiviral treatment available for use in humans. Hantaviruses belong to the order Bunyavirales with a tri-segmented negative-sense RNA genome. They encode only five viral proteins and replicate and transcribe their genome in the cytoplasm of infected cells. However, many details of the viral amplification cycle are still unknown. In recent years, structural biology methods such as cryo-electron tomography, cryo-electron microscopy, and crystallography have contributed essentially to our understanding of virus entry by membrane fusion as well as genome encapsidation by the nucleoprotein. In this review, we provide an update on the hantavirus replication cycle with a special focus on structural virology aspects.

Application of cryo-electron tomography and subtomogram averaging to determine the structure of the Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. Assembling Ebola's box The Ebola virus nucleocapsid—a protein shell—encloses, or 'encapsidates', the viral genome and acts as a scaffold for virus assembly and as a template for genome replication. John Briggs and colleagues use cryo-electron tomography to solve the structure of the nucleocapsid of the Ebola virus. They use the structures of the Ebola virus nucleocapsid within intact viruses and recombinant assemblies to propose a model for viral RNA encapsidation and accessory protein recruitment. Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever 1 . Filoviruses are within the order Mononegavirales 2 , which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein–nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein 3 , 4 , 5 , 6 , and cryo-electron microscopy of nucleocapsid 7 , 8 , 9 , 10 , 11 , 12 or nucleocapsid-like structures 11 , 12 , 13 . There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle. Enteroviruses are non-enveloped positive-sense RNA viruses that modulate cytoplasmic membranes for replication. To enlighten how enteroviruses assemble around nascent RNA genomes and get package into autophagosomes for release, Dahmane et al. perform cryo-electron tomography of poliovirus-infected cells. They find assembly intermediates that are only present on the cytosolic side of the replication compartment and provide evidence that host factor VPS34 is involved in progression of assembly intermediates.

The Varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87 kDa monomer that oligomerizes to form the pORF54 portal dodecamer. Located at a single viral capsid vertex, the portal facilitates the translocation of the newly synthesized viral genome into the preformed empty capsid. Previously described α-methylbenzyl thiourea compounds were shown to inhibit VZV DNA encapsidation, likely by targeting pORF54. In this study, drug resistant isolates were obtained via passage of VZV in increasing concentrations of one analog, Compound I (Comp I). Mutations identified in four compound resistant isolates (amino acids 48, 304, 324 and 407) all localized to a region of the portal that was predicted to interface with capsid proteins. The portal is known to undergo significant conformational changes at the portal–capsid interface during DNA encapsidation. A set of recombinant viruses was designed to reveal the chemical and physical importance of each of the resistance mutations at the portal–capsid interface, the proposed binding site of the compound series. In addition, we employed a novel complementing cell line to show that despite the presence of the portal in the virion, DNA encapsidation did not occur. We propose that a-methylbenzyl thiourea compounds perturb interactions at or near the portal–capsid interface and prevent conformational changes needed to support DNA encapsidation.

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (K d ≲ 400 nM) reside within a ~150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (μDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free μDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type μDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.

HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. ΨCES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to ΨCES to enable cotranscriptional shedding of the 5′ cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.

Summary Tomato chlorosis virus (ToCV) causes an important disease that primarily affects tomato, although it has been found infecting other economically important vegetable crops and a wide range of wild plants. First described in Florida (USA) and associated with a ‘yellow leaf disorder’ in the mid‐1990s, ToCV has been found in 35 countries and territories to date, constituting a paradigmatic example of an emergent plant pathogen. ToCV is transmitted semipersistently by whiteflies (Hemiptera: Aleyrodidae) belonging to the genera Bemisia and Trialeurodes. Whitefly transmission is highly efficient and cases of 100% infection are frequently observed in the field. To date, no resistant or tolerant tomato plants are commercially available and the control of the disease relies primarily on the control of the insect vector. Taxonomy Tomato chlorosis virus is one of the 14 accepted species in the genus Crinivirus, one of the four genera in the family Closteroviridae of plant viruses. Virion and genome properties The genome of ToCV is composed of two molecules of single‐stranded positive‐sense RNA, named RNA1 and RNA2, separately encapsidated in long, flexuous, rod‐like virions. As has been shown for other closterovirids, ToCV virions are believed to have a bipolar structure. RNA1 contains four open reading frames (ORFs) encoding proteins associated with virus replication and suppression of gene silencing, whereas RNA2 contains nine ORFs encoding proteins putatively involved in encapsidation, cell‐to‐cell movement, gene silencing suppression and whitefly transmission. Host range In addition to tomato, ToCV has been found to infect 84 dicot plant species belonging to 25 botanical families, including economically important crops. Transmission Like all species within the genus Crinivirus, ToCV is semipersistently transmitted by whiteflies, being one of only two criniviruses transmitted by members of the genera Bemisia and Trialeurodes. Disease symptoms Tomato ‘yellow leaf disorder’ syndrome includes interveinal yellowing and thickening of leaves. Symptoms first develop on lower leaves and then advance towards the upper part of the plant. Bronzing and necrosis of the older leaves are accompanied by a decline in vigour and reduction in fruit yield. In other hosts the most common symptoms include interveinal chlorosis and mild yellowing on older leaves. Control Control of the disease caused by ToCV is based on the use of healthy seedlings for transplanting, limiting accessibility of alternate host plants that can serve as virus reservoirs and the spraying of insecticides for vector control. Although several wild tomato species have been shown to contain genotypes resistant to ToCV, there are no commercially available resistant or tolerant tomato varieties to date.