Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

Viral cell-to-cell spread: Conventional and non-conventional ways.

A critical step in the life cycle of a virus is spread to a new target cell, which generally involves the release of new viral particles from the infected cell which can then initiate infection in the next target cell. While cell-free viral particles released into the extracellular environment are necessary for long distance spread, there are disadvantages to this mechanism. These include the presence of immune system components, the low success rate of infection by single particles, and the relative fragility of viral particles in the environment. Several mechanisms of direct cell-to-cell spread have been reported for animal viruses which would avoid the issues associated with cell-free particles. A number of viruses can utilize several different mechanisms of direct cell-to-cell spread, but our understanding of the differential usage by these pathogens is modest. Although the mechanisms of cell-to-cell spread differ among viruses, there is a common exploitation of key pathways and components of the cellular cytoskeleton. Remarkably, some of the viral mechanisms of cell-to-cell spread are surprisingly similar to those used by bacteria. Here we summarize the current knowledge of the conventional and non-conventional mechanisms of viral spread, the common methods used to detect viral spread, and the impact that these mechanisms can have on viral pathogenesis.

To assemble or not to assemble: The changing rules of pneumovirus transmission.

Pneumoviruses represent a major public health burden across the world. Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV), two of the most recognizable pediatric infectious agents, belong to this family. These viruses are enveloped with a non-segmented negative-sense RNA genome, and their replication occurs in specialized cytosolic organelles named inclusion bodies (IB). The critical role of IBs in replication of pneumoviruses has begun to be elucidated, and our current understanding suggests they are highly dynamic structures. From IBs, newly synthesized nucleocapsids are transported to assembly sites, potentially via the actin cytoskeleton, to be incorporated into nascent virions. Released virions, which generally contain one genome, can then diffuse in the extracellular environment to target new cells and reinitiate the process of infection. This is a challenging business for virions, which must face several risks including the extracellular immune responses. In addition, several recent studies suggest that successful infection may be achieved more rapidly by multiple, rather than single, genomic copies being deposited into a target cell. Interestingly, recent data indicate that pneumoviruses have several mechanisms that permit their transmission en bloc, i.e. transmission of multiple genomes at the same time. These mechanisms include the well-studied syncytia formation as well as the newly described formation of long actin-based intercellular extensions. These not only permit en bloc viral transmission, but also bypass assembly of complete virions. In this review we describe several aspects of en bloc viral transmission and how these mechanisms are reshaping our understanding of pneumovirus replication, assembly and spread.

Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding.

Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of antiviral strategies directed against HIV-1 infections in humans. FIV assembly results from the multimerization of a single but complex viral polypeptide, the Gag precursor. In this review, we will first give an overview of the current knowledge of the proteins encoded by the FIV pol, env, rev, vif, and orf-A genes, and then we will describe and discuss in detail the critical roles that each of the FIV Gag domains plays in virion morphogenesis. Since retroviral assembly is an attractive target for therapeutic interventions, gaining a better understanding of this process is highly desirable.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake.

This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5×109pfu/mL after dilution to 10-6pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

Production of GP64-free virus-like particles from baculovirus-infected insect cells.

The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

The Large Marseillevirus Explores Different Entry Pathways by Forming Giant Infectious Vesicles.

UNLABELLED: Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. IMPORTANCE: Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.

Scaling, crumpled wires, and genome packing in virions.

The packing of a genome in virions is a topic of intense current interest in biology and biological physics. The area is dominated by allometric scaling relations that connect, e.g., the length of the encapsulated genome and the size of the corresponding virion capsid. Here we report scaling laws obtained from extensive experiments of packing of a macroscopic wire within rigid three-dimensional spherical and nonspherical cavities that can shed light on the details of the genome packing in virions. We show that these results obtained with crumpled wires are comparable to those from a large compilation of biological data from several classes of virions.

Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

[Bolivian hemorrhagic fever].

Analysis of data of the available literature on epidemiology of Bolivian hemorrhagic fever, manifestations of human disease, biological properties of the causative agent and development carried out abroad of means and methods of diagnostics, prophylaxis and therapy of this infection that presents a potential threat for the population and economy of the Russian Federation in case of introduction of the causative agent is presented.

Virus maturation.

The formation of infectious virus particles is a highly complex process involving a series of sophisticated molecular events. In most cases, the assembly of virus structural elements results in the formation of immature virus particles unable to initiate a productive infection. Accordingly, for most viruses the final stage of the assembly pathway entails a set of structural transitions and/or biochemical modifications that transform inert precursor particles into fully infectious agents. In this chapter, we review the most relevant maturation mechanisms involved in the generation of infectious virions for a wide variety of viruses.

AcMNPV core gene ac109 is required for budded virion transport to the nucleus and for occlusion of viral progeny.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.

Simultaneous expression of recombinant proteins in the insect cell-baculovirus system: production of virus-like particles.

The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined.

Production of HIV particles is regulated by altering sub-cellular localization and dynamics of Rev induced by double-strand RNA binding protein.

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1-dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

Molecular biology of rotavirus entry and replication.

Rotavirus is a nonenveloped, double-stranded, RNA virus belonging to the Reoviridae family and is the major etiological agent of viral gastroenteritis in young children and young animals. Remarkable progress in the understanding of the rotavirus cycle has been made in the last 10 years. The knowledge of viral replication thus far acquired is based on structural studies, the expression and coexpression of individual viral proteins, silencing of individual genes by siRNAs, and the effects that these manipulations have on the physiology of the infected cell. The functions of the individual rotavirus proteins have been largely dissected; however, the interactions between them and with cell proteins, and the molecular mechanisms of virus replication, are just beginning to be understood. These advancements represent the basis for the development of effective vaccination and rational therapeutic strategies to combat rotavirus infection and diarrhea syndromes. In this paper, we review and try to integrate the new knowledge about rotavirus entry, replication, and assembly, and pose some of the questions that remain to be solved.

Cidofovir inhibits genome encapsidation and affects morphogenesis during the replication of vaccinia virus.

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus-host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20-mer peptides, covering the entire protein, HPLC-purified, (125)I-radiolabeled and tested in VERO and HeLa cell-binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54-77) and 18294 (274-308) from HPV16 L1, as well as 18312 (59-78) and 18322 (259-278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface-exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase-treated cells. Cross-linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV-16 VLP binding to HeLa cells. According to the L1- and VLP-reported structure, both peptides are close on the VLP-surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell-binding regions.

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein.

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.

Redistribution of cyclophilin A to viral factories during vaccinia virus infection and its incorporation into mature particles.

Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.