A nuclear export signal in the matrix protein of Influenza A virus is required for efficient virus replication

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.     

Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells

The surface distribution of the envelope glycoproteins of influenza, Sendai and Vesicular Stomatitis viruses was studied by immunofluorescence and immunoelectromicroscopy in infected epithelial cell monolayers, from which these viruses bud in a polarized fashion. It was found that before the onset of viral budding, the envelope proteins are exclusively localized into the same plasma membrane domains of the epithelial cells from which the virions ultimately bud: the glycoproteins of influenza and Sendai were detected at the apical surface, while the G protein of Vesicular Stomatitis virus was concentrated at the basolateral region. On the other hand, Sendai virus nucleocapsids, which can be easily identified in the cytoplasm before viral assembly, could be observed throughout the cell, not showing any preferential localization near the surface that the virions utilize for budding. These results are consistent with a model in which the asymmetric distribution of viral envelope proteins, rather than a polarized delivery of nucleocapsids, directs the polarity of viral budding. Furthermore, the asymmetric surface localization of viral glycoproteins suggests that these proteins share with intrinsic surface proteins of epithelial cells common biogenetic mechanisms and informational features or "sorting out" signals that determine their compartmentalization in the plasma membrane.     

Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import

Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.     

Introduction of a temperature-sensitive phenotype into influenza A/WSN/33 virus by altering the basic amino acid domain of influenza virus matrix protein

Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.     

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.     

Influenza B and C Virus NEP (NS2) Proteins Possess Nuclear Export Activities

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Hepatocytes traffic and export hepatitis B virus basolaterally by polarity-dependent mechanisms

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.     

Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins.

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.     

Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.     

Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus.

Black Creek Canal (BCC) virus is a newly identified hantavirus from Florida which is carried by the cotton rat (Sigmodon hispidus) and is associated with hantavirus pulmonary syndrome (HPS). We have investigated the interaction of BCC virus with polarized epithelial cells to examine whether entry and release of this virus occur at specific plasma membrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluorescence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC virus infection. In contrast, Vero C1008 cells infected basolaterally exhibited a barely detectable level of BCC virus-synthesized proteins. Titration of virus from apical and basolateral media of infected cells has demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the basolateral media. The site of BCC virus release from polarized cells is, therefore, different from that previously described for release of other members of the family Bunyaviridae and may reflect one of the determinants of hantavirus pathogenesis. In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the apical surface of polarized cells. Electron microscopy studies of the infected cells revealed evidence of BCC virus budding at the plasma membrane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Since the same site of virus assembly was recently described for Sin Nombre virus, it is likely that all of the new American hantaviruses associated with HPS utilize this same type of virus maturation.     

Polarized insertion of new membrane from a cytoplasmic reservoir during cleavage of the Drosophila embryo

Cellularization of the Drosophila embryo is a specialized form of cytokinesis that results in the formation of a polarized epithelium. The mechanisms of membrane growth during cytokinesis are largely unknown. It is also unclear whether membrane growth and polarization represent distinct processes that occur simultaneously or whether growth of the membrane is involved in the emergence of polarity. Using a combination of surface labeling and particles tracking techniques, we monitored the dynamics of marked membrane regions during cellularization. We find that the major source of membrane is intracellular, rather than in the form of a plasma membrane reservoir. Depolymerization of microtubules inhibits the export of a newly synthesized transmembrane protein from the Golgi apparatus to the plasma membrane and simultaneously blocks membrane growth. Membrane insertion occurs in a defined sequence at specific sites, first apical, then apical-lateral. Diffusion of the membrane appears insufficient to compete with the massive local insertion of new membrane. We thus identify a tightly regulated scheme of polarized membrane insertion during cellularization. We propose that such a mechanism could participate in the progressive emergence of apical-basal polarity.     

A putative nuclear function for mammalian Staufen

In addition to its role in rRNA processing and ribosome assembly, the nucleolus plays a part in the assembly of non-ribosomal ribonucleoprotein particles (RNPs) that are destined for cytoplasmic RNA delivery. Recent evidence indicates that mammalian Staufen2, a brain-specific RNA-binding protein involved in RNA localization, can--at least transiently--enter the nucleolus. Therefore, the assembly of Staufen2 into transport-competent RNPs might occur in the nucleus before their export into the cytoplasm. This could provide new insights into the mechanisms of subcellular RNA localization.     

Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.