Intracellular Virus Particles Research Articles

Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly.

Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.

Transfer of HIV-1 to Human Tonsillar Stromal Cells Following Cocultivation with Infected Lymphocytes

The susceptibility of normal human tonsillar stromal cells (HTSCs) to infection by HIV-1 was assessed using transmission electron microscopy (TEM), immunocytochemistry, and HIV-1-specific PCR analyses. Our results demonstrate that HTSCs are efficiently infected following cocultivation with the HIV-1-infected lymphoblastoid cell line GY1. Infected stromal cells contain intracellular viral particles present as free virus or associated with phagocytic vesicles. These particles express the HIV-1-specific p24 antigen as assessed by immunocytochemical analyses using an HIV-specific anti-p24 monoclonal antibody. Moreover, PCR analysis of genomic DNA isolated from particle-bearing tonsillar stromal cells identified HIV-1-specific sequences not present in either uninfected stromal cells or parental GY1 uninfected cells. The mechanism by which HIV-1 infects HTSCs does not appear to be CD4 mediated, as none of the human tonsillar stromal cell lines express CD4 as assessed by flow cytometry, immunohistochemistry, and PCR analysis. Taken together, these results demonstrate that human tonsillar stromal cells can be infected by HIV-1, and that subsequent to infection the viral genome is reverse transcribed, and integrated into the stromal cell DNA. The infection of HTSCs may contribute to HIV-1-mediated pathogenesis indirectly as a viral reservoir or directly by structural and functional modification of the lymphoid microenvironment.

Assembly of enveloped respiratory syncytial virus particles within the cytoplasm of infected Vero cells.

Electron microscopic examination of syncytia induced by a bovine respiratory syncytial virus strain in Vero cell cultures revealed the presence in the cytoplasm of assembled enveloped virus particles within inclusions of variable sizes. Moreover, budding virus particles were shown occasionally in the intracytoplasmic vesicles. These particles were filamentous in form, about 80-120 nm in diameter, variable in length, containing 12 nm diameter nucleocapsids, and looked like the extracellular particles budding at the plasma membranes. This is the first report on assembly of intracellular virus particles in cells infected by a member of the family Paramyxoviridae. Vero cell-dependent variations appear to be the factor leading to the defect in the late virus replication cycle.

St Louis encephalitis virus establishes a productive, cytopathic and persistent infection of Sf9 cells

The Sf9 cell line, commonly used for gene expression by recombinant baculovirus, has been productively infected by St Louis encephalitis (SLE) virus, a flavivirus. SLE viral infection produced a c.p.e. in the Sf9 cells characterized by giant cells and the presence of 10-fold fewer cells in the infected cultures after the first week of infection compared with uninoculated control cultures. Infected Sf9 cells expressed SLE viral antigens, and intracellular virus particles were observed by electron microscopy. Titres of cell-associated SLE virus rose slightly over an 8 week period, whereas titres of cell-free virus remained stable, suggesting that SLE virus establishes a productive and persistent infection of Sf9 cells. The SLE virus produced by the Sf9 cells could be neutralized by SLE virus-immune mouse ascitic fluid, and no evidence of escape mutants was detected. Sf9 cells persistently infected with SLE virus could be superinfected with a recombinant baculovirus and expressed recombinant antigen. The successful infection of Sf9 cells by SLE virus represents the first report of production of c.p.e. by SLE virus in insect cells under routine cell culture conditions and of the infection of Sf9 cells by a human pathogen.

An NH2-terminal peptide from the vaccinia virus L1R protein directs the myristylation and virion envelope localization of a heterologous fusion protein.

The vaccinia virus L1R gene product is a late protein destined for insertion into the envelope of intracellular virus particles. Because this protein is co-translationally modified by the addition of myristic acid to the penultimate NH2-terminal glycine residue, it was of interest to identify the modification signal within the L1R protein and to assess the relevance of myristylation to protein localization. To this end, a family of chimeric reporter genes containing 0-13 codons from the NH2 terminus of the L1R open reading frame fused in-frame to the bacterial chloramphenicol acetyltransferase gene was constructed. The encoded proteins were tested as myristylation substrates in cell-free extracts and infected cells. The results obtained in vitro and in vivo were similar and suggested that although the NH2-terminal 5 amino acids of the L1R protein were the minimum signal required to observe modification by myristate, 12 amino acids were required to obtain wild type levels of myristylation with a modulating role played by the intervening amino acid residues. Furthermore, subcellular fractionation of infected cells expressing the fusion proteins indicated that the NH2 terminus of the L1R protein was capable of targeting the fusion proteins to membrane-containing fractions only if myristylated. In particular, the myristylated fusion protein containing the first 12 amino acids of the L1R protein abutted to the chloramphenicol acetyltransferase protein was found associated with the envelope of intracellular vaccinia virus particles.

Effect of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine on the replication and morphogenesis of herpes simplex virus type 1

Treatment of African green monkey kidney cells with 1 μ/ml of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) inhibited the release of infectious herpes simplex virus type 1 (HSV-1) by more than 90%. Electron microscopic observations of HPMPC-treated monkey kidney cells demonstrated few intracellular or extracellular viral particles. The viral particles seen were without dense cores. In addition, HPMPC blocked cell fusion induced by HSV-1 in monkey kidney cells. Immunoblot analysis showed that HPMPC significantly blocked the expression of HSV-1-specific proteins. Furthermore, HPMPC inhibited the synthesis of viral DNA as determined by in situ hybridization. These results indicate that HPMPC inhibits the replication of HSV by blocking one of the events involved in DNA synthesis.

The detection of intracellular bluetongue virus particles within ovine erythrocytes

1. 1. We report here a simplified method for detecting viruses and other antigenic agents in red blood cells (RBCs). Using a nonionic detergent to prepare cytoskeletons, the interior of RBCs can be scanned rapidly using immunoelectron microscopy. 2. 2. In this study, RBCs from bluetongue (BLU) virus-infected sheep were adsorbed directly onto Formvar-coated, gold electron microscope grids. 3. 3. Cytoskeletons were prepared and then probed using a monoclonal antibody to VP 7, a structural BLU-virus protein and Protein-A gold. 4. 4. Of the ca 32,000 RBCs that were examined from BLU virus-infected sheep, 34 (0.106%) contained labelled BLU virus particles. 5. 5. No labelled particles were observed in any of ca 8000 RBCs taken prior to BLU virus inoculation of sheep. 6. 6. If the antigenic BLU virus particles (which may be viral cores) are in fact infectious, this method of sequestration of virus within RBCs could contribute to the prolonged viremia typical of this arboviral disease, which is known to occur concurrently with circulating neutralizing antibody.

IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress

A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system utilises the E. coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled. In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically required for these processes. In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be developed in a similar way.

Analysis of Nonproductive Human Immunodeficiency Virus Type 1 Infection of Human Fetal Dorsal Root Ganglia Glial Cells

Direct infection of glia by human immunodeficiency virus type 1 (HIV-1) has been suggested as one of several mechanisms responsible for the severe neurologic complications observed in both neonates and adults with the acquired immunodeficiency syndrome. We have demonstrated by protein immunoblotting analysis that HIV-1 infection of human fetal glial cells isolated from the dorsal root ganglia (DRG) of the developing human peripheral nervous system results in viral gag antigen expression with little, if any, detectable env gene products. No cytopathogenicity was evident in the infected cell population. Blot hybridization analyses indicate transient expression of the HIV-1 genome with maximum levels of virus-specific RNA being observed between 2 and 3 days postinfection and decreasing below the limits of detection by 16 days postinfection. To determine whether infection of the human fetal DRG glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase assays were performed. Direct assay of HIV-1-infected neural cell supernatants as well as exposure of permissive SupT1 cells to these HIV-1-infected neural cell supernatants resulted in no demonstrable reverse transcriptase activity in either the HIV-1-infected DRG glial cell supernatants or the SupT1 cell supernatants. Although transmission electron microscopy analyses have suggested the absence of intracellular viral particles, highly electron-dense inclusions in the cytoplasm of HIV-1-infected DRG glial cells were observed. The nature of the intracellular cytoplasmic inclusions is under current investigation. Cumulatively, these data suggest that the interaction of HIV-1 with human fetal DRG neural cells results in transient expression of the HIV genome culminating in a nonproductive infection.

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.

Analyses of the terminal sequences of west nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre-M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C protein. Priorto or during assembly of intracellular virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues 1–105, 124–290, and 291–787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells.

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum.

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.

Glucose trimming and mannose trimming affect different phases of the maturation of Sindbis virus in infected BHK cells

The roles of glucose and mannose trimming in the maturation of Sindbis virus in BHK cells have been investigated using inhibitors of glycoprotein oligosaccharide processing. In the presence of the glucosidase inhibitor N-methyl-1-deoxynojirimycin the viral glycoproteins were equipped with oligosaccharides of the composition GIc 3Man 8,9(GIcNAc) 2 and the yield of virus in the extracellular medium was reduced as a result of a block in the proteolytic cleavage of the precursor (pE2) of the E2 viral envelope glycoprotein. The mannosidase I inhibitor 1-deoxymannojirimycin (dMM) also inhibited the appearance of virus in the medium and the oligosaccharides on the viral glycoproteins had the composition Man 9(GIcNAc) 2. However, pE2 was cleaved to E2 under these conditions, and it was found that when the yield of virus from the cells and medium together was considered, there was no difference between untreated and dMM-treated cultures, suggesting the presence of intracellular virus particles in the dMM-treated cultures. When examined by electron microscopy, the dMM-treated cultures were found to contain intracellular virus particles. In addition, nucleocapsids were found lining intracellular membranes. These observations taken together with the plaque test data intimate that Sindbis virus preferentially buds from internal membranes in BHK cells treated with dMM. The results confirm the essential role of glucose trimming in the Sindbis virus-BHK cell system and suggest that the initial stages of mannose removal maybe important too.

Characterization of a second bovine rotavirus serotype

Bovine rotavirus (BRV) V 1005 was characterized by two-way cross-neutralization tests as a second serotype of BRV. Virions and inner shell particles of 65 nm and 55 nm diameter respectively, and empty capsids of 65 nm and 55 nm diameter were separated by density gradient centrifugation. Three polypeptides of molecular weight 60,000, 36,000 and 28,000 (minor protein) could be identified in the outer shell of virions and in the larger empty capsids. Inner shell particles contained three polypeptides of molecular weight 105,000, 83,000 and 43,000. Both sizes of empty capsids showed two polypeptides of molecular weight 75,000 and 55,000 not found in virions. Pulse-labelling of infected cells revealed eight major and three minor intracellular viral polypeptides. Viral polypeptides synthesis started at about 6 hours p.i. and correlated in time with double-stranded RNA synthesis. As soon as viral polypeptide synthesis was detectable, newly synthesized viral polypeptides were incorporated into intracellular viral particles. Radioactive viral polypeptides appeared without a longer lag period in extracellular viruses from 6 hours p.i. onwards.

Ultrastructural features of epithelial cell degeneration in rectal crypts of patients with AIDS.

Focal crypt epithelial cell degeneration (apoptosis) of the rectum is a characteristic pathologic feature in AIDS. The presence of apoptosis usually implies cell-mediated cytolysis, which would be an unexpected finding in this disease. We investigated the ultrastructural features of apoptosis in rectal biopsies from five AIDS patients (three homosexual males and two females with i.v. drug abuse), three heterosexual controls, and two homosexual male controls. Apoptosis was found in all AIDS patients and, to a lesser extent, in one heterosexual control. Ultrastructurally, vacuolization of several adjacent cells, leading to extrusion of cellular debris through the basal lamina and phagocytosis by macrophages, was seen. No intracellular or extracellular viral particles were detected in the regions containing apoptotic bodies, in epithelial cells near the crypt bases, in intraepithelial lymphocytes, or in macrophages. In summary, apoptosis in the rectal crypts of patients with AIDS has the same ultrastructural features as in other conditions, which suggests that its pathogenesis is related to immune rather than infectious factors. If this process occurs on a chronic basis in multiple cell types, it would promote general tissue depletion, which has been demonstrated to occur in AIDS. The presence of apoptosis in AIDS is not explained by current concepts of disease pathogenesis.

Organization of cytoskeleton elements during herpes simplex virus type 1 infection of human fibroblasts: an immunofluorescence study.

Cultured human fibroblasts showed a typical fibrillar organization of microtubules in immunofluorescence, including the vimentin type of intermediate filament as well as actin-containing microfilaments. During infection with herpes simplex virus type 1 (HSV-1), the vimentin organization was maintained whereas actin, myosin and tubulin showed a progressive association with the viral glycoproteins within juxtanuclear structures. These structures could also be revealed with fluorochrome-coupled wheat germ agglutinin. Disruption of the microtubules by demecolcine treatment or their stabilization by taxol treatment did not prevent the aggregation of viral proteins in the cytoplasm. Taxol stabilization of the microtubules allowed the juxtanuclear accumulation of the glycoproteins in HSV-infected cells whereas treatment with demecolcine led to an accumulation of the glycoproteins either in small vesicles in the cytoplasm or in the focal adhesion areas of the cells. Production of infectious intracellular virus particles was reduced in cells treated with demecolcine or with taxol before and during infection. The results of this study indicate that the normal intracellular transport and distribution of the HSV glycoproteins and the formation of infectious virus are dependent on the presence of intact microtubules.

Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment.

A 37,000-dalton polypeptide (p37K) present on purified extracellular vaccinia virus but absent from intracellular virus particles of classical morphology (G. Hiller et al., J. Virol. 39:903-913, 1981; L. G. Payne, J. Virol. 27:28-37, 1978) was further characterized. The polypeptide was only expressed in infected cells after onset of viral DNA replication. Phase partition experiments showed that it is relatively hydrophobic. Although p37K apparently is not a glycoprotein, in vivo radioisotope labeling detected tightly associated palmitic acid. Antibodies to p37K were used to monitor its distribution within infected cells at the light and electron microscopic levels. After synthesis p37K first accumulated in the Golgi region due to a tight membrane association. During progressing infection p37K-carrying membranes were used to form double-walled envelopes around brick-shaped vaccinia particles. Within these specialized vesicles vaccinia particles were moved through the cytoplasm toward the cell's surface, presumably along cellular routes for certain secretory products. Finally, single enveloped viruses were released into the extracellular space by an exocytotic process.

ハスで見出されたラブドウイルス；ハス条はんウイルス（ｌｏｔｕｓ ｓｔｒｅａｋ ｖｉｒｕｓ；ＬＳＶ）について

A previously undescribed rhabdovirus was found from East Indian lotus (Nelumbo nucifera) showing streak on rootstalks and often chlorotic or ring spots on leaves, collected at Okayama prefecture in 1977. The rhabdovirus, ca. 90×300-340nm, having helical nucleocapsid with a pitch of 4.5nm, was detected in dip, partially purified and thin-sectioned preparations. Intracellular virus particles were observed in the nucleus and cytoplasm of various cells. Nucleus of cells infected with the virus was characterized by the development of nuclear viroplasms. It suggested that the virus particles were maturated at the inner nuclear membrane and then passed through ER membrane into the cytoplasm. Since this is the first report of the virus isolate from Nymphaeaceae, as far as we know, the virus was designated lotus streak virus (LSV).

Accumulation and breakdown of RNA-deficient intracellular virus particles in interferon-treated NIH 3T3 cells chronically producing Moloney murine leukemia virus.

Interferon treatment of NIH 3T3 cells chronically infected with Moloney murine leukemia virus inhibited about 95% of virus release. This inhibition was accompanied by a three- to twofold accumulation of intracellular virions. However, this accumulation could be demonstrated only be exogenous reverse transcriptase reaction assay or radioactive labeling of the assembled viral proteins. It could not be shown by the endogenous reverse transcriptase reaction assay, which depended on endogenous viral RNA, or by labeling the encapsidated viral RNA. It was therefore evident that most of the intracellular virions accumulated in interferon-treated cells were RNA deficient. Hybridization analysis revealed that these virions were deficient of genomic viral RNA, whereas size analysis by gel electrophoresis suggested that the deficiency of 4S RNA normally packaged in Moloney murine leukemia virus was even stronger. Our data also suggested that this RNA deficiency was not due to degradation of the encapsidated RNA, but more likely to a defect in virus assembly. RNA-lacking intracellular virions were unstable; they were found to collapse before being released.

Effect of Interferon on the Formation and Release of Intracellular Virions in NIH/3T3 Cells Chronically Infected with Moloney Murine Leukemia Virus

Using transmission electron microscopy, we demonstrate in this study the existence of intracellular Moloney murine leukemia virus particles in cytoplasmic vacuoles of chronically infected NIH/3T3 cells. Interferon (IFN) treatment for 48 h resulted in a 3-fold higher accumulation of these vacuolar virions. Intracellular virions and their accumulation in IFN-treated cells could also be shown by radioactive labeling of the cells for 48 h and isolation of labeled virus particles from the cytoplasmic fraction of the cells by banding in sucrose gradient. These virions contained typical viral RNA, proteins and glycoproteins, as determined by gel-electrophoresis. Despite their higher accumulation, the formation rate of the intracellular virions, as measured by their pulse labeling for 1 h with S35-methionine, was found to be remarkably lower in IFN-treated cells. In order to elucidate this discrepancy, we followed the kinetics of the intracellular and extracellular virus appearance. We found that the inhibitory effect of IFN on the release of virus particles to the culture medium was much stronger than its effect on the formation of the intracellular virions. This finding can explain the accumulation of intracellular virus particles in IFN-treated cells despite their reduced formation rate.