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Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5\' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5\' polyA element, resulting in formation of an extended 5\' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5\' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5\' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5\' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5\' cap exposure were both poorly packaged and more efficiently translated than those that favored 5\' cap sequestration. We propose that structural plasticity of 5\' polyA and other conserved RNA elements place the 5\' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid\'s inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP\'s intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world\'s adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNA-seq profiling of miRNAs and mRNAs expressed in CD4+ T lymphocytes upon HIV-1 infection. Our results reveal significant alterations in miRNA and mRNA expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4+ T cells. The addition of miRNA-26a mimics to CD4+ T cells reduces CD59 at both the mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking out CD59 in CD4+ T cells renders both HIV-1-infected cells and progeny virions more prone to antibody-dependent CML (ADCML). The addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction in CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.