Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

Release of HSV-1 Cell-Free Virions: Mechanisms, Regulation, and Likely Role in Human-Human Transmission

Herpes simplex virus type 1, or HSV-1, is a widespread human pathogen that replicates in epithelial cells of the body surface and then establishes latent infection in peripheral neurons. When HSV-1 replicates, viral progeny must be efficiently released to spread infection to new target cells. Viral spread occurs via two major routes. In cell-cell spread, progeny virions are delivered directly to cellular junctions, where they infect adjacent cells. In cell-free release, progeny virions are released into the extracellular milieu, potentially allowing the infection of distant cells. Cell-cell spread of HSV-1 has been well studied and is known to be important for in vivo infection and pathogenesis. In contrast, HSV-1 cell-free release has received less attention, and its significance to viral biology is unclear. Here, I review the mechanisms and regulation of HSV-1 cell-free virion release. Based on knowledge accrued in other herpesviral systems, I argue that HSV-1 cell-free release is likely to be tightly regulated in vivo. Specifically, I hypothesize that this process is generally suppressed as the virus replicates within the body, but activated to high levels at sites of viral reactivation, such as the oral mucosa and skin, in order to promote efficient transmission of HSV-1 to new human hosts.

The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. We could also reveal the SPβ-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

In-depth characterization of the chikungunya virus replication cycle

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found other infection cellular activities than those usually described for the chikungunya virus replication cycle, i.e. we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area; and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Regulation of Hepatitis B Virus Virion Release and Envelopment Timing by Nucleocapsid and Envelope Interactions

Interactions between the N-terminal (assembly) domain (NTD) and the linker region of the hepatitis B virus (HBV) capsid protein and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulates covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA was further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment, and highlights the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. Importance Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases including cancer. The interactions between the HBV capsid and the large (L) envelope protein is required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.

Vpr shapes the proviral landscape and polyclonal HIV-1 reactivation patterns in cultured cells

Cell culture models suggest that the HIV-1 viral protein R (Vpr) is dispensable for latency establishment. However, whether Vpr affects the persistent proviral landscape and responsiveness to latency reversing agents (LRAs) is unclear. Here, integration site landscape, clonal dynamics, and latency reversal effects of Vpr were studied by comparing barcoded vpr+ and vpr- populations arising after infection of Jurkat cells in vitro. The results showed that individual integrant clones differed in fractions of LTR-active daughter cells: some clones gave rise to few to no LTR-active cells while for others almost all daughter cells were LTR-active. Integrant clones with at least 60% LTR-active cells (high LTR-active clones) contained proviruses positioned closer to preexisting enhancers (H3K27ac) and promoters (H3K4me3) than clones with <30% LTR-active cells (low LTR-active clones). Comparing vpr+ and vpr- populations revealed that the vpr+ population was depleted of high LTR-active clones. Complementing vpr-defective proviruses by transduction with vpr 16 days after infection led to rapid loss of high LTR-active clones, indicating that the effect of Vpr on proviral populations occurs post-integration. Comparing vpr+ and vpr- integration sites revealed that predominant vpr+ proviruses were farther from enhancers and promoters. Correspondingly, distances to these marks among previously reported intact HIV proviruses in ART-suppressed patients were more similar to those in the vpr+ pool than to vpr- integrants. To compare latency reactivation agent (LRA) responsiveness, the LRAs prostratin and JQ1 were applied separately or in combination. vpr+ and vpr- population-wide trends were similar, but combination treatment reduced virion release in a subset of vpr- clones relative to when JQ1 was applied separately, an effect not observed in vpr+ pools. Together, these observations highlight the importance of Vpr to proviral population dynamics, integration site landscapes, and responsiveness to latency reversing agents.One Sentence SummaryExpression properties and responsiveness to latency reactivation agents of individual HIV-1 proviral clones within polyclonal populations are masked by dominant clones and influenced by proviral proximity to certain epigenetic marks and by Vpr, a viral factor not previously known to affect latency and reactivation.

Human parainfluenza virus type 1 regulates cholesterol biosynthesis and establishes quiescent infection in human airway cells

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.

An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. “Complex” retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. “Simple” retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. Importance While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.

Condensate-mediated reactivation of mumps virus infection under stress

Viruses can establish both acute and persistent chronic infections, and some viruses have developed the ability to switch between the two. However, the molecular mechanisms that trigger a transition from a benign chronic infection into pathogenesis remain unknown. Here we investigated the role of the cellular stress response in provoking a chronic-to-acute transition in viral replication in a model of mumps virus infection. Using a combination of cell biology, whole-cell proteomics and cryo-electron tomography we show that stress induces phosphorylation of the disordered viral Phosphoprotein, which we suggest facilitates partitioning of the viral polymerase into preformed viral condensates, constituting the core components of the viral replication machinery. This occurs concomitantly with a conformational change in the viral nucleocapsids that exposes the viral genome and can further facilitate its replication. These changes in the viral condensate upon exogenous stress, accompanied by down-regulation of the host antiviral response, provide an environment that supports up-regulation of viral replication and virion release. Thus, we elucidate molecular and structural mechanisms of a stress-mediated switch that disrupts the equilibrium between the virome and the host in chronic infection.