José Esparza

Biochemical Evidence for the Oligomeric (Possibly Trimeric) Structure of the Major Inner Capsid Polypeptide (45K) of Rotaviruses

The major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 M-CaCl2 removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomeric form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.

Penetration and Uncoating of Rotaviruses in Cultured Cells

Early steps of replication (penetration and uncoating) of the OSU strain of porcine rotavirus were studied in MA-104 cells. After adsorption of trypsin-treated viruses at 4 degrees, followed by a shifting of the temperature to 37 degrees, particles were seen within coated pits, coated vesicles, and secondary lysosomes, indicating that virus entry occurred by receptor-mediated endocytosis, and that uncoating (removal of the outer capsid) could occur by the effect of lysosomal enzymes. This latter aspect was studied using lysosomotropic drugs (chloroquine and ammonium chloride), which were found not to inhibit rotavirus replication, indicating that the low intralysosomal pH is not responsible for virus uncoating. The effect of Ca2+ concentration on intracellular rotavirus uncoating was investigated by treating cells with the calcium ionophore A23187, to increase the intracellular Ca2+ concentration during the early stages of virus replication. Under these conditions rotavirus uncoating did not occur, suggesting that the low Ca2+ concentration in the intracellular microenvironment may be responsible for rotavirus uncoating.

VP7 serotype-specific glycoprotein of OSU porcine rotavirus: coding assignment and gene sequence.

With a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 X OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity.

Isolation, complementation and preliminary phenotypic characterization of temperature-sensitive mutants of herpes simplex virus type 2.

Abstract Eight temperature-sensitive ( ts ) mutants of herpes simplex virus type 2, isolated following mutagenesis with 5-bromodeoxyuridine, were assigned to seven nonover-lapping complementation groups (A through G). Partial phenotypic characterization of the mutants at the nonpermissive temperature (38 °) revealed the following: (1) Mutants of 3 complementation groups had a DNA − phenotype, the remaining being DNA + . (2) The DNA − mutants were deficient in DNA polymerase activity, whereas the DNA + mutants were not. (3) Two mutants, one DNA + and one DNA − , failed to induce thymidine kinase. (4) Except for one DNA − mutant, all mutants synthesized physical particles; however, only DNA + mutants synthesized enveloped particles. (5) Mutants belonging to 4 complementation groups were markedly more thermolabile than the wild-type virus, suggesting that they possessed alterations in viral structural proteins.

Preliminary characterization of virus-like particles in a mosquito (Aedes pseudoscutellaris) cell line (Mos. 61).

Electron microscopic examination of an Aedes pseudoscutellaris mosquito cell line (Mos. 61) revealed the presence of a large number of virus-like particles (VLP) in the cytoplasm of approximately 10% of the cells. These particles have a diameter of 36 nm, do not contain a lipid envelope, and have a buoyant density of 1.40 g/ml in CsCl. VLP contain DNA which appears to be single-stranded by SU nuclease assay. PAGE of the VLP demonstrates the presence of four structural polypeptides with molecular weights of 52K, 70K, 75K and 100K daltons, the smaller one being the majro polypeptide species and accounting for 71% of the the total protein mass. The A. pseudoscutellaris VLP appear to share some, but not all, of the characteristics of the genus Densovirus, family Parvoviridae. Their possible biologic importance and taxonomic location are still unclear.

Intertypic complementation and recombination between temperature-sensitive mutants of herpes simplex virus types 1 and 2.

Abstract In an attempt to identify functionally “common” and “type-specific” genes in the genomes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), intertypic complementation and recombination experiments between temperature-sensitive (ts) mutants of these viruses were undertaken. Intertypic complementation was carried out with seven HSV-1 ts mutants representing five complementation groups, and eight HSV-2 ts mutants representing five complementation groups. Efficient complementation was detected with most HSV-1 + HSV-2 ts mutant pairs, indicating extensive interchangeability of gene functions between HSV types 1 and 2. Progeny analysis of complementation yields demonstrated that complementation was symmetric; i.e., equal amounts of both HSV-1 and HSV-2 ts mutants were produced. HSV-1 wild-type virus was also able to complement HSV-2 ts mutants efficiently. Intertypic recombination experiments demonstrated that heterologous mutant pairs which failed to complement also failed to recombine efficiently.

A study on the ultrastructure of human rotavirus

Abstract The ultrastructure of human rotavirus was studied using the negative staining-carbon film technique. A new model for the human rotavirus structures is presented, based on observations made on virions and lattices formed by subviral components. In this model we proposed the existence of an inner capsid, composed of morphological subunits arranged as an open mesh, showing a regular pattern of cavities or holes, constituting the 5- and 6- coordinated units of hte virion. Morphological subunits are organizd in such a way that tow of them are always shared with each one of the neighboring holes. Analysis of selected particles suggests the existence of 162 holes formed by the icosahedral arrangement of 320 subunits, with a pattern characteristic of T = 16. Closer examination of hte morphological subunits revealed that they wre trimeric structures, giving a total of 960 structural units or protomers per virion. Even when the structure of the outer capsid could not be directly revealed by our studies, we suggest that it follows the same symmetry as the inner shell, formed by planar holey structures located on top of the inner shell subunits, resulting in the wheel shape that gave the name to the rotaviurs genus.

Enhanced Replication of Temperature-Sensitive Mutants of Herpes Simplex Virus Type 2 (HSV-2) at the Nonpermissive Temperature in Cells Transformed by HSV-2

In order to determine whether herpes simplex virus type 2 (HSV-2) genetic information present within HSV-2-transformed cells could assist the replication of temperature-sensitive (ts) mutants of HSV-2 at the nonpermissive temperature (38°), the replication of 8 ts mutants of HSV-2 was examined in normal, SV40-transformed, and HSV-2-transformed hamster cells at 38°. The growth of two DNA-positive ts mutants was significantly enhanced in two HSV-2-transformed cell lines at 38° as compared with their growth in normal cells. No enhancement of growth of any of the eight ts mutants occurred in SV40-induced tumor cells. These results suggest that resident, functional HSV genetic information in HSV-2-transformed cells assisted the replication of the mutants by complementation mechanisms.

Complementation of adeno-associated satellite viral antigens and infectious DNA by temperature-sensitive mutants of herpes simplex virus

Abstract Production of type 4 adeno-associated satellite virus (ASV) structural antigens and infectious DNA was studied using temperature-sensitive ( ts ) mutants of herpes simplex virus (HSV) types 1 and 2. Seven of the eight mutants studied were unable to synthesize HSV DNA at the nonpermissive temperature. Mutants belonging to three HSV-1 and two HSV-2 complementation groups complemented ASV antigen production while members of two other HSV-1 groups did not. Infectious satellite virus DNA was isolated from the HSV-1 ts -1 complementing system.

Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela

Abstract The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983–February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4–6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.