Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in Escherichia coli

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn²⁺. Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY–p-nitroanilide was found to be cleaved by the 2A protease with a k_cat/K_m ratio of ~335 M⁻¹s⁻¹, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.     

RNA Signals in Entero- and Rhinovirus Genome Replication

The VPg-linked, plus-stranded RNA genomes of entero- and rhinoviruses contain very different 5′ and 3′ terminal regions which harbor signals for RNA replication. The terminal cloverleaf-like structure of the 5′-nontranslated region (5′NTR) is known to be required for plus-strand RNA synthesis. Genetic evidence suggest that two stem-loop structures and the poly(A) tail of the 3′NTR have a function in minus-strand synthesis. All of the nonstructural viral proteins, and possibly also some cellular polypeptides, are believed to be involved in RNA replication. RNA synthesis is initiated on a poly(A) template and involves uridylylation of VPg to yield VPgpU(pU). This precursor is likely to serve as primer for the RNA polymerase 3D^polduring both minus- and plus-strand RNA synthesis.     

Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 × 10⁸ particles per 10⁶ cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.     

Preliminary Observations Pertaining to Polyadenylation of Rhinovirus RNA

Human rhinovirus type 14 contained polyadenylated RNA. Virus growth in HeLa cells was inhibited by cordycepin or polyuridilic acid and stimulated by polyadenylic acid. Polyadenylic acid also reversed cordycepin inhibition of virus-induced cytopathology of infected HeLa cells.     

Human Rhinovirus Type 14:Human Immunodeficiency Virus Type 1 (HIV-1) V3 Loop Chimeras from a Combinatorial Library Induce Potent Neutralizing Antibody Responses against HIV-1

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.     

Properties of Light Particles Produced During Growth of Type 4 Adeno-Associated Satellite Virus

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm³ after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm³. These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm³ were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 × 10⁶ daltons, compared to 1.4 × 10⁶ for the complete satellite DNA molecule.     

RNA Dependent DNA Polymerase in C Type Particles from Normal Rat Thymus Cultures

THE isolation and characterization of C type particles released from normal rat thymus cell cultures has been described^{1, 2}. The detection of RNA dependent DNA polymerase activity in these C type particles is reported here. Some properties of the enzymatic activities found in C type particles isolated from normal rat thymus cultures are described and compared with DNA polymerase found in isolated Moloney leukaemia virus (MLV) particles purified from the growth media of rat thymus cultures infected chronically with MLV². The sensitivity to actinomycin D is different in the two kinds of C type particles, suggesting that the DNA polymerases are not identical.     

Analysis of Human T-Cell Leukemia Virus Type 1 Particles by Using Cryo-Electron Tomography

The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.     

Rhinovirus RNA Polymerase: Products and Kinetics of Appearance in Human Diploid Cells

The kinetics of appearance of an RNA-dependent RNA polymerase activity obtained from human embryo lung cells infected with rhinovirus type 2 have been followed by analysis of the RNA synthesized by the polymerase preparation in vitro. Little single-stranded RNA was synthesized and the proportion of replicative intermediate to replicative form was over threefold greater than obtained in vivo. The polymerase activity in vivo declined in the presence of cycloheximide showing that continued protein synthesis was necessary to maintain RNA replication.     

Antigenic Determinants of Infective and Inactivated Human Rhinovirus Type 2

Treatment of human rhinovirus type 2 (HRV 2) virions at pH 5, at 56 C or in 2 M urea, produces one or both of two types of subviral particles. These subviral particles sediment at 135S or at 80S and both share what have been designated as C-antigenic determinants; the determinants of native virions have been designated D. These sets of determinants have been contrasted by the techniques of immunodiffusion, complement fixation, and serum blocking, and the results indicate that many or most of the D-determinants are lost in the conversion to C antigenicity. Some of the HRV 2 C-determinants also react, in immunodiffusion and in complement fixation tests, with antisera produced against HRV 1A virions. The inverse reaction has also been detected by complement fixation. Purified natural top component (NTC) of HRV 2 contains C- and, to a lesser extent, D-determinants. The D-determinants of NTC are also, like those of virions, lost upon treatment at pH 5. These results are discussed in terms of a conformational model for the D- to C-antigenic conversion.     

Virus Particles in Apparently Healthy Peregrinus maidis

Virus-like particles were found in apparently healthy Peregrinus maidis (Ashm.) in Venezuela. The particles were observed in the salivary gland, intestine, mycetome, adipose tissue, ovary, and hemolymph. In the cells, the particles occurred in the cytoplasm singly, in groups, free or within vesicles, and in hexagonally arranged crystals. In P. maidis from Hawaii such particles were not found. However, in these insects, single particles or crystals were observed after injecting suspensions of intestines from P. maidis from Venezuela. The particles were not observed in insects feeding on plants soiled with excretions from particle-containing P. maidis. Particles in organs of insects or in pellets of intestines were polygonal and showed a weakly contrasted envelope and a highly contrasted core. The particles had a diameter of 54 ± 9 mμ. They are believed to represent a Peregrinus virus causing latent infection.     

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E^rns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E^rns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of E^rns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, E^rnsE1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of E^rns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase.     

Hepatitis C Virus: Assembly and Release of Virus Particles

Hepatitis C virus is a blood-borne virus that typically establishes a chronic infection in the liver, which often results in cirrhosis and hepatocellular carcinoma. Progress in understanding the complete virus life cycle has been greatly enhanced by the recent availability of a tissue culture system that produces infectious virus progeny. Thus, it is now possible to gain insight into the roles played by viral components in assembly and egress and the cellular pathways that contribute to virion formation. This minireview describes the key determining viral and host factors that are needed to produce infectious virus.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.