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Results for “viral genome uncoating”

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At least 19 recordsLinked to original sources
PubMed2006

The inner tegument promotes herpes simplex virus capsid motility along microtubules in vitro.

After viral fusion, capsids of the neurotropic herpes simplex virus are transported along microtubules (MT) to the nuclear pores for viral genome uncoating, nuclear transcription and replication. After assembly and egress from the nucleus, cytosolic capsids are transported to host membranes for secondary envelopment or to the axon terminal for further viral spread. Using GFP-tagged capsids, Cy3-labelled MT and cytosol, we have reconstituted viral capsid transport in vitro. In the presence of ATP, capsids moved along MT up to 30 microm. Blocking the function of dynactin, a cofactor of dynein and kinesin-2, inhibited the transport. Removing outer tegument proteins from the capsids increased in vitro motility. In contrast, capsids isolated from infected nuclei that were devoid of inner as well as outer tegument proteins showed little interaction with dynein and its cofactor dynactin. Our data suggest that the inner tegument of alphaherpesviruses contains viral receptors for MT motors.

Adenosine Triphosphate↗
PubMed1999

Conformational changes in the hepatitis A virus capsid in response to acidic conditions.

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.

Acids↗
PubMed2026

RNA-repelling Anionic Clusters in Human Rhinovirus Cooperate with Cationic Residues to Promote Virion Assembly and Restrain RNA Release.

Research on virus nucleic acid-protein interactions is important to understand infection and guide antiviral drug design. In previous studies we showed that the human rhinovirus (RV) genomic RNA is organized as a dodecahedral cage formed by 30 RNA duplex elements anchored to capsid concavities. We showed also that capsid-RNA duplex interactions include conserved tryptophans, neutral polar residues, and many positively charged residues that promote virion assembly and restrain RNA release by stabilizing the negatively charged RNA duplex structure. The present study expands our understanding of the capsid-RNA duplex interface in RV by addressing the structural and functional roles of conserved patches of negatively charged capsid residues interposed between each RNA duplex and its binding site at the capsid inner surface. The initial hypothesis was that electrostatic repulsion between anionic residues and RNA phosphates would lead to functional effects opposite to those previously found for cationic residues that can electrostatically attract RNA phosphates. In fact, those anionic residues do not oppose, but act together with cationic residues at the RNA duplex binding sites to promote virion assembly and restrain RNA release. Cryogenic electron microscopy analysis showed that negatively charged residues at the capsid-RNA duplex interfaces have a different structural role than positively charged residues, even though they all play similar functional roles. A tentative model is discussed to explain the functional effects of the complex distribution of negative and positive electrostatic potential found at capsid-RNA duplex interfaces in RV.

capsid↗
PubMed2004

Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway.

The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.

Actin Cytoskeleton↗
PubMed2024

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far. Antiviral drug design may benefit from an understanding of the role during the infectious cycle of the interactions in the virion between the capsid and the viral nucleic acid. The genomic RNA inside the human RV virion forms a dodecahedral cage made of 30 double-stranded RNA elements that interact with equivalent sites at the capsid inner wall. RNA dodecahedral cages also occur in distantly related insect and plant viruses. However, the functional role(s) of the interactions between any dodecahedral cage and the capsid remained to be established. Here we describe an extensive structure-function mutational analysis of the capsid-RNA dodecahedral cage interface in the RV virion, to dissect the role of the interactions between the capsid and the cage-forming RNA duplexes in: (i) infection by RV; (ii) virus biological fitness; (iii) virion assembly; (iv) virion stability; and (v) viral RNA uncoating. The results reveal that the capsid-bound dsRNA dodecahedral cage in the human RV virion is a multifunctional structural element. Two structurally overlapping subsets of RNA duplex-capsid interactions promote virus infectivity and biological fitness by respectively facilitating virion assembly or restraining the untimely, unproductive uncoating of the viral RNA genome. These results provide new insights into virion morphogenesis and genome uncoating, and have implications for antiviral drug design.

RNA, Viral↗
PubMed2006

[Host factors that regulate the intercellular dynamics of HIV-1 genome during the early phase of infection].

An interplay or battle between virus and its host has been observed within a single cell. Upon an infection with retroviruses including human immunodeficiency virus type 1 (HIV-1), the viral genome is subjected to several processes that include uncoating, reverse transcription of the viral genomic RNA into a cDNA copy, transport of this cDNA into the nucleus, and integration of the cDNA into the host chromosome. Antiretroviral restriction factors such as TRIM5 alpha and APOBEC3G have been recently identified. In addition, nuclear membrane protect host chromosomal DNA against incoming viral genome. For successful retroviral infection, viral genome must overcome these cellular barriers to establish proviral state, in which viral cDNA was stably integrated into host chromosomal DNA. In this review, I would summarize the host factors that regulate the intercellular dynamics of HIV-1 genome during the early phase of infection, especially focusing on factors interacting with HIV-1 integrase and the preintegration complex.

APOBEC-3G Deaminase↗
PubMed2000

Image reconstructions of helical assemblies of the HIV-1 CA protein.

The type 1 human immunodeficiency virus (HIV-1) contains a conical capsid comprising approximately 1,500 CA protein subunits, which organizes the viral RNA genome for uncoating and replication in a new host cell. In vitro, CA spontaneously assembles into helical tubes and cones that resemble authentic viral capsids. Here we describe electron cryo-microscopy and image reconstructions of CA tubes from six different helical families. In spite of their polymorphism, all tubes are composed of hexameric rings of CA arranged with approximate local p6 lattice symmetry. Crystal structures of the two CA domains were 'docked' into the reconstructed density, which showed that the amino-terminal domains form the hexameric rings and the carboxy-terminal dimerization domains connect each ring to six neighbours. We propose a molecular model for the HIV-1 capsid that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal 'defects'.

Capsid↗
PubMed1984

Early events in the replication of parvovirus LuIII.

Purified infectious LuIII virions characterized for their biological and physicochemical properties were used to analyse early events in the interaction of an autonomous parvovirus with its host cell. It could be shown that adsorption and penetration of LuIII virus are rapid processes which, under optimal conditions, can be completed within 30 to 60 minutes of incubation at 37 degrees C. Uncoating of the viral genome is initiated in the cytoplasm and is continued after the virus has been transported to the nucleus. None of these processes appeared to depend on cell cycle-specific helper function(s). However, the first step in viral replication--formation of viral parental RF-DNA consisting of covalently linked complementary strands--evidently could only be achieved in late S-phase of the cell cycle. No evidence was obtained which would support the hypothesis that viral DNA must be integrated into the cellular genome to allow for the initiation of viral DNA synthesis.

Biological Transport↗
PubMed1984

Infection of a Chlorella-like alga with the virus, PBCV-1: ultrastructural studies.

Ultrastructural studies revealed that the virus, PBCV-1, adsorbs to the surface of the Chlorella-like green alga NC64A and enzymatically digests a portion of the host cell wall. The viral DNA is then released into the interior of the cell leaving an empty capsid on the surface. Thus uncoating of the viral genome occurs at the surface of its host. PBCV-1 also adsorbs to and digests the host wall of either heat-killed, methanol-extracted, or purified cell wall fragments.

Chlorella↗
PubMedDate not supplied

Myristic acid is coupled to a structural protein of polyoma virus and SV40.

In the lytic cycle of papova viruses, both uncoating of the viral genome after infection and assembly of functional virions take place in the cell nucleus. The mechanisms by which newly internalized virions are targeted to the nucleus and viral DNA encapsidated into particles are poorly understood. Although the major capsid protein VP1 is involved in endocytosis, and largely defines virion structure, the functions of the minor proteins VP2 and VP3 have remained obscure. Here we show that VP2 from both polyoma virus and simian virus 40 (SV40) is covalently linked to myristic acid; this is the first report of a myristylated protein in the nucleus and of a fatty acid being important in the structure of a nonenveloped virus. We consider the implications of this unusual modification on encapsidation and suggest that VP2 may be a scaffolding protein for virion assembly.

Amino Acid Sequence↗
PubMed1986

Inhibition of Banzi viral RNA synthesis in BHK-21 cells by Banzi virion matrix protein.

Treatment of BHK-21 cells with purified matrix (M) protein isolated from Banzi virions resulted in decreased production of Banzi virus and in reduced levels of virus-specific positive-strand and negative-strand RNA. The M protein had no effect on the uncoating of the viral genome. This inhibition was virus-specific since the replication of unrelated togaviruses in BHK-21 cells was not affected. Purified M protein inhibited the in vitro activity of the Banzi viral RNA polymerase; prior treatment of the M protein with either trypsin or antiviral serum blocked this inhibition.

Animals↗
PubMed2005

Genome prediction of putative genome-linked viral protein (VPg) of astroviruses.

Positive-sense single-stranded RNA (+ssRNA) viruses replicate by uncoating the RNA genome for translation to provide viral proteins essential for genome replication and the production of new viral particles. The viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently. The synthesized proteins include viral RNA-dependent RNA polymerase (RdRP), viral genome-linked protein (VPg), and a helicase. VPg is covalently attached to the genomic form of +ssRNA viruses. Helicases and NTPase unwind the RNA before replication. VPg and helicases have been identified in +ssRNA families, however, the presence of VPg and helicase in the Astroviridae, another +ssRNA family, has not been fully elucidated. Computational tools were utilized to provide sequence analysis evidence for the presence and genomic location of astrovirus VPg and helicase. HMMER program v2.1.1 was used to build Hidden Markov Model (HMM) profile for calicivirus VPg to search for conserved motifs in the astrovirus genome. We performed phylogenetic analysis of two genomic regions of astroviruses and caliciviruses (encoding the RdRP and VPg). We identified a putative VPg coding region in astrovirus. This region was located in open reading frame 1a (ORF1 a) and included sites with high sequence similarity to the VPg coding regions of Caliciviridae, Piconaviridae, and Potyviridae. A region encoding a putative astrovirus helicase identified conserved motifs only with pestivirus helicase sequences. Sequence analysis and comparison to other +ssRNA viruses supports the presence of VPg in the Astroviridae. Further laboratory analysis will be necessary to confirm these findings.

Amino Acid Sequence↗
PubMedDate not supplied

Virus synthesis and replication: reovirus vs. vaccinia virus.

The strategies with which two viral genomes that consist of double-stranded nucleic acid express themselves in infected cells are compared. The reovirus genome comprises ten segments of double-stranded RNA, each of which is, in essence, a gene. Each is transcribed into plus-stranded RNA which has two functions: to serve as messenger RNA for the synthesis of the ten reovirus "primary" proteins, and to serve as template for the synthesis of minus-strands with which they remain associated, thereby giving rise to progeny double-stranded RNA. One of the most fascinating unsolved features of the reovirus multiplication cycle is the nature of the mechanism that ensures that each progeny virus particle contains a complete set of the ten individual genome RNA segments. The vaccinia virus genome is a linear molecule of double-stranded DNA which possesses sizable terminal redundancies (up to 7 percent, depending on the strain). The vaccinia virus multiplication cycle can be divided into a well-defined early and late period. During the early period, infecting virus particles are first uncoated to cores within which some 40-50 percent of the viral genome is transcribed. These cores are then uncoated further to naked viral DNA, a process that is mediated by protein(s) translated from the "core" messenger RNA. The overall transcription pattern in highly complex and is regulated both at the transcriptional as well as at the translational level. The most profound program changes occur at the time when DNA replication begins, when the transcription of "early" messenger RNAs, some of which are translated into "early" enzymes, gives way to that of "late" messenger RNAs, most of which are translated into structural virus components.

DNA, Viral↗
PubMed2006

Viral interactions with the cytoskeleton: a hitchhiker's guide to the cell.

The actin and microtubule cytoskeleton play important roles in the life cycle of every virus. During attachment, internalization, endocytosis, nuclear targeting, transcription, replication, transport of progeny subviral particles, assembly, exocytosis, or cell-to-cell spread, viruses make use of different cellular cues and signals to enlist the cytoskeleton for their mission. Viruses induce rearrangements of cytoskeletal filaments so that they can utilize them as tracks or shove them aside when they represent barriers. Viral particles recruit molecular motors in order to hitchhike rides to different subcellular sites which provide the proper molecular environment for uncoating, replicating and packaging viral genomes. Interactions between subviral components and cytoskeletal tracks also help to orchestrate virus assembly, release and efficient cell-to-cell spread. There is probably not a single virus that does not use cytoskeletal and motor functions in its life cycle. Being well informed intracellular passengers, viruses provide us with unique tools to decipher how a particular cargo recruits one or several motors, how these are activated or tuned down depending on transport needs, and how cargoes switch from actin tracks to microtubules to nuclear pores and back.

Animals↗
PubMed2003

Mechanisms of hepatitis C virus infection.

Hepatitis C virus (HCV) is the major causative agent of chronic non-A, non-B hepatitis. The life cycle of HCV is largely unknown because a reliable culture system has not yet been established. HCV presumably binds to specific receptor(s) and enters cells through endocytosis, as do other members of Flaviviridae. The viral genome is translated into a precursor polyprotein after uncoating, and viral RNA is synthesized by a virus-encoded polymerase complex. Progeny viral particles are released into the luminal side of the endoplasmic reticulum and secreted from the cell after passage through the Golgi apparatus. Understanding the mechanisms of HCV infection is essential to the development of effective new therapies for chronic HCV infection. Several host membrane proteins have been identified as receptor candidates for HCV. Recent advances using pseudotype virus systems have provided information surrounding the initial steps of HCV infection. An HCV RNA replicon system has been useful for elucidating the replication mechanism of HCV. In this review, we summarize our current understanding of the mechanisms of HCV infection and discuss potential antiviral strategies against HCV infection.

Hepacivirus↗
PubMed1994

Viral gene products and replication of the human immunodeficiency type 1 virus.

The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus.

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.

Capsid Proteins↗