Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Low-Fidelity Assembly of Influenza A Virus Promotes Escape from Host Cells.

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here, we address these questions by developing a strain of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using this strain, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. This phenotypic variability contributes to virus survival during environmental challenges, including exposure to antivirals. Complementing genetic adaptations that act over larger populations and longer times, this "low-fidelity" assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.

Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit.

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

Unveiling the DHX15-G-patch interplay in retroviral RNA packaging.

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.

The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription.

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Zn2+-dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses.

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.

NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles.

Hepatitis C virus NS5A is a multifunctional phosphoprotein comprised of three domains (DI, DII and DIII). DI and DII have been shown to function in genome replication, whereas DIII has a role in virus assembly. We previously demonstrated that DI in genotype 2a (JFH1) also plays a role in virus assembly, exemplified by the P145A mutant which blocked infectious virus production. Here we extend this analysis to identify two other conserved and surface exposed residues proximal to P145 (C142 and E191) that exhibited no defect in genome replication but impaired virus production. Further analysis revealed changes in the abundance of dsRNA, the size and distribution of lipid droplets (LD) and the co-localisation between NS5A and LDs in cells infected with these mutants, compared to wildtype. In parallel, to investigate the mechanism(s) underpinning this role of DI, we assessed the involvement of the interferon-induced double-stranded RNA-dependent protein kinase (PKR). In PKR-silenced cells, C142A and E191A exhibited levels of infectious virus production, LD size and co-localisation between NS5A and LD that were indistinguishable from wildtype. Co-immunoprecipitation and in vitro pulldown experiments confirmed that wildtype NS5A domain I (but not C142A or E191A) interacted with PKR. We further showed that the assembly phenotype of C142A and E191A was restored by ablation of interferon regulatory factor-1 (IRF1), a downstream effector of PKR. These data suggest a novel interaction between NS5A DI and PKR that functions to evade an antiviral pathway that blocks virus assembly through IRF1.

Assembly of infectious Kaposi's sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer.

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its ß-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem-loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem-loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

Defining the Assembleome of the Respiratory Syncytial Virus.

During respiratory syncytial virus (RSV) particle assembly, the mature RSV particles form as filamentous projections on the surface of RSV-infected cells. The RSV assembly process occurs at the / on the cell surface that is modified by a virus infection, involving a combination of several different host cell factors and cellular processes. This induces changes in the lipid composition and properties of these lipid microdomains, and the virus-induced activation of associated Rho GTPase signaling networks drives the remodeling of the underlying filamentous actin (F-actin) cytoskeleton network. The modified sites that form on the surface of the infected cells form the nexus point for RSV assembly, and in this review chapter, they are referred to as the RSV assembleome. This is to distinguish these unique membrane microdomains that are formed during virus infection from the corresponding membrane microdomains that are present at the cell surface prior to infection. In this article, an overview of the current understanding of the processes that drive the formation of the assembleome during RSV particle assembly is given.

Selective packaging of HIV-1 RNA genome is guided by the stability of 5' untranslated region polyA stem.

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

Using T4 genetics and Laemmli's development of high-resolution SDS gel electrophoresis to reveal structural protein interactions controlling protein folding and phage self-assembly.

One of the most transformative experimental techniques in the rise of modern molecular biology and biochemistry was the development of high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis, which allowed separation of proteins-including structural proteins-in complex mixtures according to their molecular weights. Its development was intimately tied to investigations of the control of virus assembly within phage-infected cells. The method was developed by Ulrich K. Laemmli working in the virus structural group led by Aaron Klug at the famed Medical Research Council Laboratory for Molecular Biology at Cambridge, UK. While Laemmli was tackling T4 head assembly, I sat at the next bench working on T4 tail assembly. To date, Laemmli's original paper has been cited almost 300,000 times. His gel procedure and our cooperation allowed us to sort out the sequential protein-protein interactions controlling the viral self-assembly pathways. It is still not fully appreciated that this control involved protein conformational change induced by interaction with an edge of the growing structure. Subsequent efforts of my students and I to understand how temperature-sensitive mutations interfered with assembly were important in revealing the intracellular off-pathway aggregation processes competing with productive protein folding. These misfolding processes slowed the initial productivity of the biotechnology industry. The article below describes the scientific origin, context, and sociology that supported these advances in protein biochemistry, protein expression, and virus assembly. The cooperation and collaboration that was integral to both the Laboratory for Molecular Biology culture and phage genetics fields were key to these endeavors.

Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly.

Most viruses undergo a maturation process from a weakly self-assembled, noninfectious particle to a stable, infectious virion. For herpesviruses, this maturation process resolves several conflicting requirements: (i) assembly must be driven by weak, reversible interactions between viral particle subunits to reduce errors and minimize the energy of self-assembly, and (ii) the viral particle must be stable enough to withstand tens of atmospheres of DNA pressure resulting from its strong confinement in the capsid. With herpes simplex virus 1 (HSV-1) as a prototype of human herpesviruses, we demonstrated that this mechanical capsid maturation is mainly facilitated through capsid binding auxiliary protein UL25, orthologs of which are present in all herpesviruses. Through genetic manipulation of UL25 mutants of HSV-1 combined with the interrogation of capsid mechanics with atomic force microscopy nano-indentation, we suggested the mechanism of stepwise binding of distinct UL25 domains correlated with capsid maturation and DNA packaging. These findings demonstrate another paradigm of viruses as elegantly programmed nano-machines where an intimate relationship between mechanical and genetic information is preserved in UL25 architecture. IMPORTANCE The minor capsid protein UL25 plays a critical role in the mechanical maturation of the HSV-1 capsid during virus assembly and is required for stable DNA packaging. We modulated the UL25 capsid interactions by genetically deleting different UL25 regions and quantifying the effect on mechanical capsid stability using an atomic force microscopy (AFM) nanoindentation approach. This approach revealed how UL25 regions reinforced the herpesvirus capsid to stably package and retain pressurized DNA. Our data suggest a mechanism of stepwise binding of two main UL25 domains timed with DNA packaging.

An ESCRT/VPS4 Envelopment Trap To Examine the Mechanism of Alphaherpesvirus Assembly and Transport in Neurons.

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)-tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFP-VPS4-EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. IMPORTANCE Alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), are pathogens of the nervous system. They replicate in the nerve cell body and then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells; however, key aspects of how these viruses travel along axons remain controversial. Here, we test two alternative mechanisms for transport, the married and separate models, by blocking envelope assembly, a critical step in viral egress. When we arrest formation of the viral envelope using a mutated component of the cellular ESCRT apparatus, we find that entry of viral particles into axons is blocked in some types of neurons but not others. This approach allows us to determine whether envelope assembly occurs prior to entry of viruses into axons or afterwards and, thus, to distinguish between the alternative models for viral transport.

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny.

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.