Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.     

Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.     

The packaging signal of influenza viral RNA molecules.

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5'-bulged promoter structure, as caused by the central unpaired residue A10 in its 5' branch. Upon insertion of two uridine residues in the 3' branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3'-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5'-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5'A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5' bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.     

Competitive incorporation of homologous gene segments of influenza a virus into virions

By using two reporter protein-encoding virus-like RNAs derived from identical viral RNA (vRNA) segments, we assessed their incorporation efficiency into single progeny virions. Most plaques formed by the recombinant viruses that were generated in cells positive for both reporter genes expressed only one or the other protein. These results suggest that two virus-like RNAs encoding different reporter proteins compete for incorporation into virions, and individual influenza virions incorporate single, but not multiple, copies of homologous vRNA segments.     

Crucial role of CA cleavage sites in the cap-snatching mechanism for initiating viral mRNA synthesis

In viral cap-snatching, the endonuclease intrinsic to the viral polymerase cleaves cellular capped RNAs to generate capped fragments that are primers for viral mRNA synthesis. Here we demonstrate that the influenza viral polymerase, which is assembled in human cells using recombinant proteins, effectively uses only CA-terminated capped fragments as primers for viral mRNA synthesis in vitro. Thus we provide the first in vitro system that mirrors the cap-snatching process occurring in vivo during virus infection. Further, we demonstrate that when a capped RNA substrate contains a CA cleavage site, the functions of virion RNA (vRNA) differ from those previously described: the 5' terminal sequence of vRNA alone is sufficient for endonuclease activation, and the 3' terminal sequence of vRNA functions solely as a template for mRNA synthesis. Consequently, we are able to identify the vRNA sequences that are required for each of these two separable functions. We present a new model for the influenza virus cap-snatching mechanism, which we postulate is a paradigm for the cap-snatching mechanisms of other segmented, negative-strand and ambisense RNA viruses.     

Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus ((TMV)MP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that (TMV)MP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of (TMV)MP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.     

'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs

Many viruses exploit cellular polarity to constrain the assembly and release of progeny virions to a desired surface. Influenza virus particles are released only from the apical surface of epithelial cells and this polarization is partly owing to specific targeting of the viral membrane proteins to the apical plasma membrane. The RNA genome of the virus is transcribed and replicated in the nucleus, necessitating nuclear export of the individual ribonucleoprotein (RNP) segments before they can be incorporated into budding virus particles. We show that the process of polarized virus assembly begins in the nucleus with the RNPs adopting a novel asymmetric distribution at the inner nuclear membrane prior to their export to the cytoplasm. The viral nucleoprotein, the major protein component of RNPs, displays the same polarized intranuclear distribution in the absence of other influenza virus components, suggesting the existence of a hitherto unrecognized polarity within the mammalian cell nucleus.     

Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes

At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3' end of the coding region and 80 nucleotides at the 5' end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.     

Dynamics of filamentous viral RNPs prior to egress

The final step in the maturation of paramyxoviruses, orthomyxoviruses and viruses of several other families, entails the budding of the viral nucleocapsid through the plasma membrane of the host cell. Many medically important viruses, such as influenza, parainfluenza, respiratory syncytial virus (RSV) and Ebola, can form filamentous particles when budding. Although filamentous virions have been previously studied, details of how viral filaments bud from the plasma membrane remain largely unknown. Using molecular beacon (MB)-fluorescent probes to image the viral genomic RNA (vRNA) of human RSV (hRSV) in live Vero cells, the dynamics of assembled viral filaments was observed to consist of three primary types of motion prior to egress from the plasma membrane: (i) filament projection and rotation, (ii) migration and (iii) non-directed motion. In addition, from information gained by imaging the 3D distribution of cellular vRNA, observing and characterizing vRNA dynamics, imaging vRNA/Myosin Va colocalization, and studying the effects of cytochalasin D (actin depolymerizing agent) exposure, a model for filamentous virion egress is presented.     

The 5′ Cap of Tobacco Mosaic Virus (TMV) Is Required for Virion Attachment to the Actin/Endoplasmic Reticulum Network During Early Infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)‐labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3‐virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co‐injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell‐to‐cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3‐vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin‐dependent RNA movement. The 5′ methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5′ cap failed to form granules and was degraded in the cytoplasm. Removal of the 3′ untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual‐labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER‐bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.     

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed.     

Macromolecular interactions in the assembly of HIV and other retroviruses

A critical phase in the infection cycle of HIV and other retroviruses is the assembly of new infectious virus particles. This process requires complex but coordinated targeting of capsid precursor proteins, virus genomic RNA and viral glycoproteins to a common assembly site on the plasma membrane. Domains within the capsid precursor proteins define the route taken to the plasma membrane and direct the process of virus budding. However, in order for the assembled virus to be infectious, viral glycoproteins, replicative enzymes and genomic RNA must also be included. The mechanisms by which this complex of interactions occur are discussed in this chapter.     

Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions

A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.     

HIV-1 assembly is initiated by p17 matrix protein

HIV-1 matrix protein (MA) is multifunctional structural protein located on N-terminus of Gag precursor p55 and responsible for its transport to plasma membrane, the site of virus assembly. Here, it has been shown that MA is cleaved from Gag precursor at early stage of the virus infection and participates in virus assembly. MA is transported into the nuclei wherein it associates with viral RNA (vRNA). The MA-vRNA complex is transported to plasma membrane. Mutant MA which lost its membranotropic signal does not reach plasma membrane and MA-vRNA complex remains in the nuclei and cytoskeleton. Thus, MA seems to deliver vRNA from the nuclei to plasma membrane through cytoskeleton initiating virus assembly.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.     

流感病毒在脂筏上组装与出芽

流感病毒造成的季节性流行性疾病给全世界带来沉重的健康负担.近年来,甲型流感病毒的变种H5N1、H7N9给各国带来了很大危害.流感病毒属于正黏附病毒科,它的遗传物质由多个节段的负链RNA组成,其组装和出芽剪切生殖是一个涉及到多种病毒因子,多步骤、复杂的生化过程.流感病毒会使用宿主的细胞膜上的"脂筏"区域作为病毒出芽位点.首先病毒的两种糖蛋白NA蛋白、HA蛋白会在脂筏区域聚集,造成脂筏区膜变形弯曲,并且发动出芽的过程.接着,流感病毒基质蛋白M1的C端与HA、NA结合,其自身在脂筏区域开始多聚化并使膜向外弯曲形成原始病毒体的内部结构,接着招募病毒的核糖核蛋白复合物(VRNP)与M2蛋白,使组装的过程进一步完成.最后,M2蛋白会富集在原始病毒体的底部,完成膜的剪切和病毒体的释放.     

Two modes of herpesvirus trafficking in neurons: membrane acquisition directs motion

Alphaherpesvirus infection of the mammalian nervous system is dependent upon the long-distance intracellular transport of viral particles in axons. How viral particles are effectively trafficked in axons to either sensory ganglia following initial infection or back out to peripheral sites of innervation following reactivation remains unknown. The mechanism of axonal transport has, in part, been obscured by contradictory findings regarding whether capsids are transported in axons in the absence of membrane components or as enveloped virions. By imaging actively translocated viral structural components in living peripheral neurons, we demonstrate that herpesviruses use two distinct pathways to move in axons. Following entry into cells, exposure of the capsid to the cytosol resulted in efficient retrograde transport to the neuronal cell body. In contrast, progeny virus particles moved in the anterograde direction following acquisition of virion envelope proteins and membrane lipids. Retrograde transport was effectively shut down in this membrane-bound state, allowing for efficient delivery of progeny viral particles to the distal axon. Notably, progeny viral particles that lacked a membrane were misdirected back to the cell body. These findings show that cytosolic capsids are trafficked to the neuronal cell body and that viral egress in axons occurs after capsids are enshrouded in a membrane envelope.     

HIV-1 assembly initiated by p17 matrix protein

HIV-1 matrix protein (MA) is a multifunctional structural protein located on the N-terminus of Gag precursor p55 and is responsible for its transport to the plasma membrane, the site of virus assembly. In the present paper, it has been shown that MA is cleaved from Gag precursor at an early stage of the virus infection and participates in virus assembly. MA is transported into the nuclei wherein it associates with viral RNA (vRNA). The MA-vRNA complex is transported to the plasma membrane. Mutant MA, which lost its membranotropic signal, does not reach the plasma membrane and MA-vRNA complex remains in the nuclei and cytoskeleton. Thus, MA seems to deliver vRNA from the nuclei to plasma membrane through the cytoskeleton, initiating virus assembly.     

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import: IMPLICATIONS FOR HIV-1 GENE EXPRESSION*

Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.