Sukla Basak

Effects of hexose starvation and the role of sialic acid in influenza virus release

We previously reported that growth of influenza virus in the presence of cytochalasin B (CB), a drug that disrupts microfilaments and blocks hexose transport, yields particles with glycoproteins that are heterogeneous and unlabeled by [3H]glucosamine. When the virus was grown in glucose-free medium, we observed reduced virus titers similar to those produced by CB. In contrast, treatment of cells with cytochalasin D (CD) and dihydrocytochalasin B (H2CB), drugs which are known to inhibit microfilament function without affecting hexose transport, did not cause a reduction in virus titers or a change in the electrophoretic mobility of viral glycoproteins. Partial inhibition of glycosylation of viral glycoproteins resulting from either CB-induced inhibition of hexose transport or from glucose starvation resulted in the formation of aggregates of virions on cell surfaces. These aggregates can be dissociated by exogenous neuraminidase. Under these conditions the virions contained a functional hemagglutinin glycoprotein (HA) but an inactive neuraminidase glycoprotein (NA) which was not able to cleave sialic acid, the HA receptor, from viral glycoproteins, or from cellular glycoproteins and glycolipids. Neuraminidase treatment of membrane fractions of CB-treated cells did not cause a shift in the electrophoretic mobility of HA or in the gel elution profile of HA glycopeptides obtained after extensive pronase digestion from HA synthesized in glucose-free medium. These findings suggest that sialic acid is not present on labeled glycoproteins in either of these preparations. We obtained evidence that the sialic acid to which HA binds when NA is inactive is on glycoproteins and glycolipids of cellular origin. Our results support the idea that even when NA is functional, sialylated cellular components impede influenza virus release.

Infectious entry pathway for canine parvovirus.

We have investigated whether canine parvovirus (CPV) infection involves a low pH-dependent entry pathway. The effects of two lysosomotropic bases, NH4Cl and chloroquine, on CPV entry were studied by immunofluorescence and ultrastructural and biochemical methods. In the presence of these reagents, input virions appear to accumulate in large vacuoles. Ultrastructural studies indicated that uptake of virions takes place predominantly in small uncoated vesicles that appear to fuse with larger vesicles. In the presence of NH4Cl, virions accumulate in the latter structures and their uncoating appears to be prevented. Viral DNA as well as antigen synthesis were found to be significantly inhibited in the presence of these reagents. In addition, inhibition of viral DNA and antigen synthesis appeared to be most extensive when NH4Cl was present from 30 min preinfection, whereas no significant inhibition was observed when the cells were treated after 2 hr postinfection. Thus, the results indicate that CPV requires exposure to low pH in an endosomal compartment to initiate a productive infection.

Studies on the role of glycosylation in the functions and antigenic properties of influenza virus glycoproteins

The biological and antigenic roles of glycosylation were investigated in the influenza hemagglutinin (HA) glycoprotein using the glycosylation inhibitor tunicamycin (TM). Under conditions where only the nonglycosylated form of HA was detected by immunoprecipitation and gel electrophoresis, the migration of glycoproteins to the cell surface was observed by immunofluorescence using either monospecific or monoclonal antibody to the HA polypeptide. Analysis of the surface fluorescence in TM-treated infected cells by a fluorescence-activated cell sorter (FACS) showed that all cells exhibited fluorescence in the complete absence of glycosylation. The relative amount of HA antigen on cell surfaces was found to be reduced by only 30-40% in TM-treated cells, and this reflected a similar reduction in intracellular synthesis. Electron microscopic studies using ferritin labeling also demonstrated that the nonglycosylated HA glycoprotein was present in significant amounts on surfaces of infected cells. Virions with nonglycosylated glycoproteins were purified, and were found to have an approximate 30-fold decrease in both hemagglutinin and neuraminidase specific activities. The possible role of oligosaccharides in antigenic variation among various H1N1 strains was investigated. Immunoprecipitation reactions involving five different monoclonal antibodies and five antigenic variants of A/USSR/90/77 revealed no major antigenic differences between the glycosylated and nonglycosylated forms of HA.

Preliminary X-ray crystallographic analysis of canine parvovirus crystals☆

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).

The complete nucleotide sequence of an infectious clone of porcine parvovirus, strain NADL-2

The complete nucleotide sequence of an infectious clone of porcine parvovirus, strain NADL-2, was determined. The nucleotide sequence organization of the viral genome was found to be similar to that of the other autonomous parvoviruses, such as canine parvovirus and minute virus of mice.

Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase

We have analyzed the pronase-derived glycopeptides of the hemagglutinin glycoproteins expressed from SV40 vectors carrying cloned cDNA copies of the HA gene and of HA isolated from influenza virions (A/Jap/305/57). The glycopeptides derived from he HA glycoprotein obtained from cloned genes were heterogeneous, ranging in size from 3800 to 2800 daltons. Upon treatment with neuraminidase, sialic acid was released from the glycopeptides and their size was reduced to 2900-2400 daltons. However, under the same conditions, no sialic acid was detected in the virion HA. The presence of sialic acid was confirmed by monosaccharide analysis of the HA glycoprotein derived from products of cloned genes. These results support the idea that during replication of influenza virus, the viral neuraminidase cleaves sialic acid from the HA glycoprotein in infected cells.

Expression of SV40 receptors on apical surfaces of polarized epithelial cells.

We have investigated the interaction of SV40 virions with polarized monkey kidney epithelial cells. Virions were tagged with biotin to facilitate their detection and were found to retain full infectivity. When polarized Vero C1008 cells were incubated with biotinylated virions followed by a strepavidin-rhodamine conjugate, distinct cell populations were identified which expressed very different levels of SV40 receptors. The parental Vero C1008 cells yielded three types of cell clones which exhibited low, moderate, or predominantly high levels of SV40 binding. Virus-binding assays to each of these clones as well as to parental Vero C1008 cells indicated that the level of SV40 receptor expression is cell-cycle-dependent. The cellular receptors for influenza A virus (WSN strain) were also found to be distributed heterogeneously on polarized epithelial cells. In contrast, in several types of nonpolarized cells, SV40 receptors were found to be uniformly distributed over the monolayer. SV40 binding was not found to correlate with HLA expression on Vero C1008 cells or other cell types. Also in contrast to SV40 receptor expression, which is restricted to the apical domain, HLA was found to be distributed on both apical and basolateral domains of Vero C1008 cells.

Nucleotide sequence analysis of the capsid genes and the right-hand terminal palindrome of porcine parvovirus, strain NADL-2

The genome of the porcine parvovirus, strain NADL-2, has been cloned and the sequence of map units 28 to 100, which is 3670 bp in length and contains the capsid coding regions and the right-hand terminal palindrome, has been determined. The sequence shows extensive homology with other parvoviruses such as MVM, H-1, CPV, and FPV in the capsid coding region. Given the information available from these sequences, the regulatory and capsid coding regions for PPV have been proposed and the amino acid sequences of capsid proteins compared. The right end, which has a 188-nucleotide-long imperfect palindromic sequence, has an organization similar to that of other parvoviruses and the homologies in this region with other parvoviruses correspond to the homologies observed in the rest of the respective genomes.

Host cell-dependent lateral mobility of viral glycoproteins☆

The lateral mobility of viral envelope proteins on the plasma membranes of infected cells is an important factor in both virus assembly and pathogenesis. The envelope glycoproteins of measles and human parainfluenza virus are mobile on the surfaces of infected HeLa cells and undergo lateral redistribution in the presence of specific antibody, forming unipolar caps. In contrast, no such redistribution was observed with influenza virus hemagglutinin (HA) or vesicular stomatitis virus (VSV) G glycoproteins on infected HeLa cell surfaces. However, the HA and G glycoproteins were both found to be mobile in the plasma membrane of CV-1 cells, or human or murine peritoneal macrophages. These results indicate that host cell-dependent as well as virus-specific factors are involved in determining viral glycoprotein mobility. No significant differences in the patterns of synthesis of influenza or VSV viral proteins were found in the various cell types examined. The HA and G proteins, when expressed from vaccinia virus recombinants, were each found to be immobile in HeLa cells and mobile in CV-1 cells, thus indicating that the host cell-dependent differences in mobility are an intrinsic property of each viral glycoprotein molecule and not the result of interaction with other viral components. It is suggested that the association of viral glycoproteins with either the cytoskeleton or membrane-associated cellular proteins may be related to the observed differences in lateral mobility.

STRUCTURE OF THE SPIKE GLYCOPROTEIN OF INFLUENZA C VIRUS

Posttranslational cleavage of the precursor glycoprotein gp88 of influenza C virus results in two subunit glycoproteins, gp65 and gp30, each of which exist in two molecular forms. Influenza C glycoproteins were isolated from purified influenza C virions by selective solubilization with Triton X-100 or octylglucoside. Only preparations obtained with octylglucoside showed hemagglutinating activity. Tryptic peptide analysis of the three species of viral glycoproteins, gp88, gp65 and gp30, revealed that gp30 and gp65 are distinct; when the peaks resolved for the two subunit glycoproteins are superimposed, the pattern corresponding to that of gp88 is obtained. The two molecular forms of gp65 have an identical polypeptide backbone as shown by tryptic peptide analysis. The N-termini of gp88 as well as gp65 were resistant to sequential Edman degradation. The gp30 terminal sequence contains a preponderance of hydrophobic residues and shows homology with the corresponding sequence of the HA^ subunit of influenza A viruses, except for an additional N-terminal glycine residue. The glycoprotein spikes on the surface of influenza C virions were found in regular hexagonal arrays, which appear to involve lateral interaction between the glycoprotein molecules, since the spikes sometimes maintain their arrangement in a network upon release from the viral membrane by limited proteolytic digestion, or upon spontaneous disruption of the viral membrane. In infected cells, closely packed surface projections were observed on crescent shaped outfoldings of the plasma membrane, where virus maturation is occurring. The fact that, in most cases, no nucleocapsids can be seen in such regions suggests that nucleocapsids may not be required to initiate outfolding of the plasma membrane in the budding process of influenza C virions.