Formation Of Infectious Virus Research Articles

Inhibition of adenovirus infection with protease inhibitors

The effect of a series of cysteine and serine protease inhibitors was tested on the growth of human adenovirus type 2 in tissue culture. In accordance with the nature of the adenovirus protease, only the cysteine protease inhibitors were effective in significantly reducing the production of infectious virus. Addition of the inhibitors to the medium 18 h after infection gave IC 50 of 30, 40 and 80 nM with N-ethylmaleimide, leupeptin and E64c, respectively. Several lines of evidence suggest that inhibition of infectious virus formation operated through the inhibition of the viral protease rather than cellular toxicity: (a) the yield of physical particles declined only 4–5-fold, while that of infectious virus declined 3–7 orders of magnitude, (b) these particles contained unprocessed precursor proteins and (c) pulse-chase experiments showed that the inhibitors prevented the efficient processing of viral precursor proteins. We conclude that the cysteine protease inhibitors efficiently depress the formation of infectious adenovirus by inhibiting the viral protease.

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.

Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ. In the stably HBV-transfected HepG 2.2.15 cells and in HBV-infected HepG2 cells, NBDNJ suppressed secretion of HBV particles and caused intracellular retention of HBV DNA. The secretion of subviral particles was less affected. These data suggest that inhibitors of oligosaccharide trimming may be useful for antiviral therapy of hepatitis B and for the study of the intracellular transport of the viral glycoproteins.

Brefeldin A affects West Nile virus replication in Vero cells but not C6/36 cells

A fungal metabolite brefeldin A (BFA) was used to study virus-host interaction in glycoprotein processing in West Nile virus-infected Vero and C6/36 cells. The results indicated that as little as 1 μg/ml of BFA resulted in complete breakdown in the Golgi organelle in infected Vero cells. This led to modifications of the glycoproteins which could not be efficiently used in infectious virion formation. In contrast, as much as 10 μg/m of BFA in culture medium did not affect either glycoprotein formation or production of infectious particles in C6/36 cells. The results showed that in Vero cells, the transport of glycoproteins to the Golgi apparatus is important in West Nile virus infection. It also showed that BFA could be used as a tool to understand further the trafficking of glycoprotein from the ER to Golgi in flavivirus infection in Vero cells.

Replacement of His 23 by Cys in a zinc finger of HIV-1 NC p7 led to a change in 1H NMR-derived 3D structure and to a loss of biological activity

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which is necessary for the formation of infectious virions, contains two zinc fingers of the Cys-X 2-Cys-X 4-His-X 4-Cys form. To elucidate the importance of this particular motif, well conserved in retroviruses and retroelements, we substituted the histidine residue by a cysteine in the first zinc binding domain 13VKCFNCGKEGHTARNCRA 30. The structures of the mutated and native zinc complexed peptides were studied by two-dimensional 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution. The nuclear Overhauser effects were used as constraints to determine the solution structures using DIANA software followed by AMBER energy refinement. The results show that native and mutant peptides fold into non-identical three-dimensional structures, probably accounting for the loss of retrovirus infectivity following the His-Cys point mutation.

The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.

Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions

The neuraminidase (NA) of influenza viruses cleaves sialic acids from receptors, prevents self-aggregation and facilitates release of virus during budding from host cells. Although the structure and function of the globular head of the influenza virus NA has been well studied, much less is known about the stalk of the NA, the region between the viral membrane and the globular head. Applying a reverse genetics system, we altered the stalk of the influenza A/WSN/33 virus NA by making deletions, insertions and mutations in this region of the gene. Our data show that the length of the NA stalk can be variable. Deletions of up to 28 amino acids and insertions of up to 41 amino acids in the stalk region did not abolish formation of infectious progeny virus. The data also indicate that the cysteine at position 76 is essential for formation of infectious virus, and that deletions beyond the cysteine did not result in infectious virus. Interestingly, shortening of the length of the stalk region by 28 amino acids resulted in a virus with a markedly reduced growth rate in MDCK cells as compared to that in MDBK cells. An insertion of 41 extra amino acids into the stalk did not significantly interfere with viral growth in MDCK or MDBK cells, which suggests that the stalk region would tolerate the introduction of long foreign sequences.

Characterization of two structurally novel HIV-1 protease inhibitors identified by rational selection

The human immunodeficiency virus (HIV-1), associated with the AIDS (acquired immunodeficiency syndrome) epidemic, encodes an aspartyl protease that is essential for polyprotein processing in the virus (Navia et al., 1989). It has been demonstrated that inactivation of the protease either catalytically or by an inhibitor prevents infectious virion formation (Kohl et al., 1988; Darke et al., 1989). The acquired knowledge of key molecular interactions occurring between inhibitors and aspartyl proteases, as well as the structural similarities between HIV-1 protease and human renin was used to rationally select candidates for HIV-1 screening from the pool of analogs designed as renin inhibitors. A minimal number of chosen compounds were tested in an HIV-1 protease assay system. Two structurally novel peptides emerged as potent enzymatic protease inhibitors. This study highlights the selection process and characterizes the antiviral properties of the two novel analogs.

Involvement of membrane traffic in the replication of poliovirus genomes: Effects of brefeldin A

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449–451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2A pro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.

Site-directed mutations in sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and El appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

A viable mouse polyomavirus mutant without immortalizing or transforming activities

The polyomavirus mutant, dl1041, has a 375-base pair deletion. It removes most of the sequences that are unique to rodent polyomaviruses and encodes part of the large and middle T-antigens. The mutant was conditionally viable, although both the immortalizing and transforming functions of the T-antigens produced by this mutant were found to be defective. However, the dl1041 mutant was found to be capable of DNA replication in rapidly growing mouse 3T6 cells. In contrast, dl1041 DNA synthesis could not be detected in serum-deprived mouse 3T3 cells. In these cells, the low efficiency of dl1041 DNA replication could be attributed to deficiencies in both large and middle T-antigen, suggesting a link between the mitogenic and oncogenic activities of these proteins. Transfection of growing mouse 3T6 cells with dl1041 DNA resulted in the formation of infectious virus, demonstrating that the dl1041 mutant is able to complete an infection cycle. The ability to activate the viral late promoter in trans was retained by the dl1041 mutant large T-antigen, suggesting that immortalization and trans-activation of the late promoter represent two distinct activities of the protein. An essential element of the immortalizing activity in the large T-antigen polypeptide chain appeared to be in a segment consisting of amino acid residues 136–184, since the dl1041 deletion abolished the activity and the 184 amino acid residue N-terminal dl1354 fragment of large T-antigen retained the activity.

Structure and assembly of bacteriophage PRD1, an Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5′ ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infectious virus.

Sequence analysis and acute pathogenicity of molecularly cloned SIVSMM-PBj14

The PBj14 isolate of simian immunodeficiency virus from sooty mangabey monkeys (SIVSMM-PBj14) is the most acutely pathogenic primate lentivirus so far described, always causing fatal disease in pig-tailed macaques (Macaca nemestrina) within 8 days of inoculation. As a first step in identifying viral genes and gene products that influence pathogenicity, the SIVSMM-PBj14 genome was amplified by the polymerase chain reaction as 5' and 3' genomic halves of 5.1 and 5.8 kilobases, respectively, and molecularly cloned. DNA sequence analysis revealed a high degree of conservation with other SIVs, except for a 22-base-pair duplication in the enhancer region of the viral long terminal repeat which included a second binding site for the transcription factor NF-kappa B. Of six genomic halves examined, four contributed to the formation of infectious virus that induced acute disease and death in pig-tailed macaques as early as 6 days post-inoculation, with pathology, disease syndromes and kinetics indistinguishable from those induced by the uncloned isolate. To our knowledge this is the first example of acute immunodeficiency disease induced by a molecularly defined lentivirus. Furthermore, the molecularly cloned SIVSMM-PBj14 viruses share with the uncloned virus cytopathicity for mangabey CD4+ cells, a property that may correlate with their observed pathogenicity in vivo.

The effect of ammonium chloride on the multiplication of herpes simplex virus type 1 in Vero cells

The multiplication of herpes simplex virus type 1 (HSV-1) in Vero cells is inhibited by ammonium chloride. The formation of infectious virus was inhibited immediately after the addition of the agent into the culture fluid and was restored by removal of the agent. Although neither viral DNA replication nor nucleocapsid formation were affected by the addition of ammonium chloride at 4 h postinfection, the agent markedly inhibited the formation of enveloped particles and completely the formation of infectious progeny virus. These results indicate that one of the effects of ammonium chloride on the multiplication of HSV-1 is the inhibition of envelopment of viral nucleocapsids. In addition, the envelopment of HSV-1 nucleocapsids was inhibited immediately after the addition of monensin into the culture fluid. These findings suggest the importance of acidic pH of an intracellular compartment in the envelopment of HSV-1.

Quantitative studies on the maturation process of herpes simplex virus type 1 in vero cells

We examined the time courses of viral DNA synthesis, the formation and envelopment of viral nucleocapsids, and the formation of infectious progeny virus in Vero cells infected with herpes simplex virus type 1 (HSV-1). The results showed that the formation of nucleocapsids coincided with the appearance of enveloped particles as well as of infectious progeny virus, although the synthesis of viral DNA took place approximately 2 h prior to the beginning of nucleocapsid formation. These results indicate that the rate-limiting step in the virogenesis of HSV-1-infected cells is the encapsidation of viral DNA and that the enveloped virus is formed immediately after the formation of nucleocapsids and is infectious without any further processing of virion constituents.

Mechanism of suppression by bovine serum on growth of infectious bursal disease virus.

The mechanism of suppression of plaque formation of infectious bursal disease virus (IBDV) by calf serum (CS) was investigated. Suppressor in CS was absorbed by incubation of the serum with chicken embryo fibroblast (CEF), indicating the attachment of suppressor to CEF. The amount of IBDV that was able to absorb to CEF decreased when the cells were pretreated with CS. Suppression of IBDV plaque formation was also noted when CS was added to agar overlay medium, indicating that the serum suppressed the spread of the virus infection to the surrounding cells. The suppressive effect of CS was not due to a direct inactivation (neutralization) of IBDV. The results indicated that the suppressor in CS attaches to the surface of CEF and probably occupies the virus receptors on the cells, therefore blocking the absorption of the IBDV and consequently, leading to the suppression of plaque formation by the virus.

Bovine rotavirus maturation is a calcium-dependent process.

Bovine rotavirus-infected MA-104 cells maintained in the presence and absence of CaCl2 displayed cytopathic effects (cpe) distinct from each other. Lysates of calcium-free cultures were unable to induce cpe in subsequent passages in MA-104 cells, an observation reflected by the demonstration that virus titers of such lysates were drastically reduced. The minimum concentration of CaCl2 in the growth medium required to maintain maximum virus yield was determined to be approximately 0.17 mM. The period of calcium-dependency for infectious virus formation was between 6 and 12 hr postinfection at 37 degrees, a time corresponding to the entire log phase of virus growth. Viruses produced in the absence of calcium were found to be exclusively incomplete single-shelled particles (D particles), as determined by cesium chloride density gradient analysis, electron microscopy, and SDS-polyacrylamide gel electrophoresis. Subsequent examination of virus-specified proteins in infected cells revealed that there was a reduction in the level of the major outer capsid protein (42K) in the absence of calcium. Thus, total inhibition of mature virus production under this condition could be due to the combined effect of reduced production of the 42K protein and incomplete assembly of the virus.

New agents active against African swine fever virus.

Actinobolin, atropine, carrageenan, megalomycin C, suramin, and tetracenomycin C were tested for their activity against African swine fever virus replication. Both viral inhibitory potency and cytotoxicity were investigated. Megalomycin C, suramin, atropine, and carrageenan exhibited significant activity. Megalomycin C was the most active of the four agents with respect to the concentration of compound that blocked the formation of infectious virus by 50%. Suramin was the next most active agent in this respect, but because of its lower cytotoxicity, it had the most favorable therapeutic index.

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.