Extracellular Virus Production Research Articles

The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus

The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.

Dehydroepiandrosterone, epiandrosterone and synthetic derivatives inhibit Junin virus replication in vitro

In the present paper the in vitro antiviral activity of dehydroepiandrosterone (DHEA), epiandrosterone (EA) and 16 synthetic derivatives against Junin virus (JUNV) replication in Vero cells was studied. DHEA and EA caused a selective inhibition of the replication of JUNV and other members of the Arenaviridae family such as Pichinde virus and Tacaribe virus. The compounds were not virucidal to cell-free JUNV. The impairment of viral replication was not due to an inhibitory effect of the steroids on virus adsorption or internalization. An inhibitory effect of the compounds on JUNV protein synthesis and both intracellular and extracellular virus production was demonstrated. A partial inhibitory action on cell surface expression of JUNV glycoprotein G1 was also detected on DHEA- and EA-treated cultures. Like DHEA and EA, three compounds obtained from EA by chemical synthesis showed selectivity indexes higher than ribavirin, the only antiviral compound that has shown partial efficacy against arenavirus infections.

Activity of the Anti-Orthopoxvirus Compound ST-246 against Vaccinia, Cowpox and Camelpox Viruses in Cell Monolayers and Organotypic Raft Cultures

The potential use of variola virus as a biological weapon has renewed efforts in the development of antiviral agents against orthopoxviruses. ST-246 [4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-di oxo-4,6-ethenocycloprop [f]isoindol-2(1 H)-yl)-benzamide] is an anti-orthopoxvirus compound active against several orthopoxviruses including vaccinia virus (VV), cowpox virus (CPV), camelpox virus (CMLV), ectromelia virus (ECTV) and variola virus in cell culture. The compound has been shown to inhibit the release of extracellular virus by targeting the F13L W protein and to protect mice from W, CPV and ECTV orthopoxvirus-induced disease. The antiviral activity of ST-246 was assessed against extracellular and intracellular W, CPV and CMLV production in human embryonic lung (HEL) fibroblasts and primary human keratinocyte (PHK) cell monolayers, as well as in three-dimensional raft cultures. ST-246 inhibited preferentially the production of extracellular virus compared with intracellular virus production in HEL and PHK cells (for W) and in PHK cells (for CMLV). In organotypic epithelial raft cultures, ST-246 at 20 microg/ml inhibited extracellular W and CMLV production by 6 logs, whereas intracellular virus yield was reduced by 2 logs. In the case of CPV, both extracellular and intracellular virus production were completely inhibited by ST-246 at 20 microg/ml. Histological sections of the infected rafts, treated with increasing amounts of drug, confirmed the antiviral activity of ST-246: the epithelium was protected and there was no evidence of viral infection. Electron microscopic examination confirmed the absence of intracellular enveloped virus forms in W-, CPV- and CMLV-infected cells treated with 10 microg/ml of ST-246. These data indicate that ST-246 is a potent anti-orthopoxvirus compound; the mode of inhibition is dependent on the virus and cell type.

An Orally Bioavailable Antipoxvirus Compound (ST-246) Inhibits Extracellular Virus Formation and Protects Mice from Lethal Orthopoxvirus Challenge

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

Trafficking mechanism of west Nile (Sarafend) virus structural proteins

Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin.

Insect cells and their potential as stabilization barriers for DNA of multiple and single nucleopolyhedroviruses against ultraviolet-B-simulated sunlight inactivation.

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

Involvement of vacuolar proton ATPase in Junin virus multiplication.

The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.

Measles virus spread between neurons requires cell contact but not CD46 expression, syncytium formation, or extracellular virus production.

In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46(+) mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46(+) neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46(+) and CD46(-) neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.

Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines.

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID50 titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to AcMNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401 + 10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401 + 10% FBS (37.8 x 10% TCID50/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4 x 10(6) TCID50/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9 x 10(6) TCID50/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.

The Complete Genome Sequence of Shope (Rabbit) Fibroma Virus

We have determined the complete DNA sequence of the Leporipoxvirus Shope fibroma virus (SFV). The SFV genome spans 159.8 kb and encodes 165 putative genes of which 13 are duplicated in the 12.4-kb terminal inverted repeats. Although most SFV genes have homologs encoded by other Chordopoxvirinae, the SFV genome lacks a key gene required for the production of extracellular enveloped virus. SFV also encodes only the smaller ribonucleotide reductase subunit and has a limited nucleotide biosynthetic capacity. SFV preserves the Chordopoxvirinae gene order from S012L near the left end of the chromosome through to S142R (homologs of vaccinia F2L and B1R, respectively). The unique right end of SFV appears to be genetically unstable because when the sequence is compared with that of myxoma virus, five myxoma homologs have been deleted (C. Cameron, S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D. Willer, D. Evans, and G. McFadden, 1999, Virology 264, 298–318). Most other differences between these two Leporipoxviruses are located in the telomeres. Leporipoxviruses encode several genes not found in other poxviruses including four small hydrophobic proteins of unknown function (S023R, S119L, S125R, and S132L), an α 2,3-sialyltransferase (S143R), a protein belonging to the Ig-like protein superfamily (S141R), and a protein resembling the DNA-binding domain of proteins belonging to the HIN-200 protein family S013L). SFV also encodes a type II DNA photolyase (S127L). Melanoplus sanguinipes entomopoxvirus encodes a similar protein, but SFV is the first mammalian virus potentially capable of photoreactivating ultraviolet DNA damage.

Effect of temperature oscillation on insect cell growth and baculovirus replication

Temperature oscillation can enhance cell viability of sf9 insect cells and baculovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture. The optimal oscillation range was 24 to 28 degreesC. At this temperature oscillation, the viability of uninfected and infected sf9 cells can be maintained much longer than at 28 degreesC. Although the rate of virus infection was a little low at 24 to 28 degreesC, the final cell infectivity was similar to that at a constant temperature of 28 degreesC. The production of OB was increased from 13.4 to 17.4/cell in stationary culture and from 13.9/cell to 18.1/cell in suspension culture. The titer of ECV was increased from 87 to 114 PFU/cell in stationary culture and from 79 to 114 PFU/cell in suspension culture.

Phosphorylation of DHBV Pre-S: Identification of the Major Site of Phosphorylation and Effects of Mutations on the Virus Life Cycle

Four potential serine/threonine phosphorylation sites [(S/T)-P motif], designated P1–P4, on the pre-S protein of duck hepatitis B virus (DHBV) have been mutated. Mutants include single (P2, P3, P4) and double amino acid substitutions (P1+P2, P3+P4) and one with all four sites mutated (4P). Serine at position 118 (P3) was identified as the major site of phosphorylation by Western blotting and radioimmunoprecipitation afterin vitrocell labeling with [35S]methionine or [33P]orthophosphate. Mutant virions generated by transfection of LMH cells were infectious bothin vitroin duck hepatocyte primary cultures andin vivoin Pekin ducks. Intracellular relaxed circular (RC) and covalently closed circular (ccc) DNA syntheses were not affected by the P3 mutation or even the quadruple mutant. Extracellular virus production was slightly increased when the P3 site was mutated. CsCl gradient centrifugation showed no clear difference between mutant and wild-type virus with respect to the ratios of enveloped virus and nucleocapsid particles in hepatocyte culture supernatants. Trypsin or V8 protease digestion with or without NP-40 indicated that phosphorylation of the pre-S domain is not involved in determining the transmembrane topology of DHBV large protein. This phenotypic analysis indicates that DHBV pre-S phosphorylation has no apparent effect on DHBV replication and formation of mature viral particles in duck hepatocyte primary culture and does not affect infectivity in ducklings.

Dual therapy with IL-12 and IFNα provides a novel approach to the management of chronic viral disease

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

Replication of Helicoverpa zea Nuclear Polyhedrosis Virus in Homologous Cell Lines Grown in Serum-Free Media

A parental cell line, BCIRL-HZ-AM1 (HZAM1) derived from Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) pupae and one of its clones BCIRL-HZAM1-B3 (HZ1B3), were grown as attached cells in two commercial insect serum-free media, EX-CELL 400 and SF-900, and one modified vertebrate serum-free medium, MCM1. A serum-containing medium, TC199MK, in which the cell lines were normally propagated, was used as control. Cell doubling times for HZAM1 in EX-CELL 400, SF-900, and TC199-MK, were 27, 28, and 30 hr, respectively; in contrast, there was negligible growth in MCM1. The shortest cell doubling time for HZ1B3 in serum-free medium was 25 hr, as observed in EX-CELL 400. Other cell doubling times were: 35 hr in TC199-MK, 38 hr in MCM1, and 45 hr in SF-900. Total extracellular virus production of H. zea nuclear polyhedrosis virus (HzSNPV)-infected HZAM1 cells was similar in all media (3.0-6.3 × 105 PFU/ml). Extracellular virus titers for HZ1B3 in EX-CELL 400 and SF-900 were similar (3-4 × 105 PFU/ml) but less than those produced in HZAM1. For HZ1B3, the lowest extracellular virus titer was recorded in MCM1 (0.8 × 105 PFU/ ml) and the highest in TC199-MK (12 × 105 PFU/ml). Production of HzSNPV occlusion bodies (OB) by HZAM1 cells differed slightly between the media (2.9-6.2 × 107 OB/ml). For HZ1B3 cells, OB production was similar in TC199-MK, EX-CELL 400, and SF-900 (3.6-6.0 × 107 OB/ml), but considerably lower in MCM1 (1.0 × 107 OB/ml). Occlusion bodies from both infected cell lines grown in serum-free media and in TC199-MK were equally infectious for H. zea larvae.

Genetic Analyses of Temperature-Sensitive Mutations in Baculovirus Late Expression Factors

Two temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were characterized with respect to late and very late gene expression. Cells infected with ts317 or ts538 at the nonpermissive temperature revealed slightly reduced intracellular levels of late structural gene products including gp64 envelope, vp39 capsid, and p6.9 DNA binding proteins. However, with the exception of gp64, there was a dramatic reduction in extracellular levels of the structural proteins, suggesting that assembly or transport of extracellular virions was impaired. Electron microscopy confirmed the dramatic effect the mutations exhibited on viral morphogenesis. The ts538 mutation was physically mapped to the 60-65 mu region and DNA sequence analysis identified a single nucleotide alteration converting Leu to Phe in amino acid 105 of the late expression factor called lef-4. The mutation was positively correlated with the temperature-sensitive phenotype by analysis of DNA from ts + revertants generated by marker rescue. Transient expression assays confirmed that lef-4 was required for activation of the late vp39 gene in virus infected cells. The defect in activation of the ts317 and ts538 polyhedrin genes could be overcome by coinfection with helper virus indicating that the mutations affected functions involved in activating the very late class of baculovirus genes as well. These results demonstrated a clear functional differentiation between genes regulating viral DNA replication which was normal in the mutant infected cells and those regulating late gene expression and extracellular virus production which were defective in these same infected cells.

Biological and Molecular Characteristics of Human Herpesvirus 7: In Vitro Growth Optimization and Development of a Syncytia Inhibition Test

Two isolates of human herpesvirus 7 (HHV-7) were recovered from phytohemagglutinin-activated peripheral blood mononuclear cells of a patient with chronic fatigue syndrome and of a healthy blood donor. A genetic polymorphism between the two isolates was detected by Southern blot analysis using a novel HHV-7 genomic clone (pVL8) as a probe. We developed optimized conditions for the in vitro propagation of HHV-7 by using enriched populations of activated CD4 + T lymphocytes derived from normal peripheral blood, resulting in the production of high-titered extracellular virus (>10 6 cell culture infectious doses/ml). Bona fide syncytia formation was documented both in normal CD4 + T lymphocytes and in the Sup-T1 CD4 + T-cell line following infection with high-titered HHV-7. To identify neutralizing antibodies to HHV-7, a syncytia-inhibition test was developed. Variable titers of syncytia-neutralizing antibodies were detected in all the human sere tested, thus confirming the high prevalence of HHV-7 in the human population.

Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence.

Human immunodeficiency virus type 1 (HIV-1) displays both interstrain and intrastrain genetic variability. Virus populations with extensive microheterogeneity have been defined as swarms or quasispecies. Many of the genomes within HIV-1 swarms appear to be defective in one or more genes required for viral replication. It is unclear to what extent defective viruses play a role in the process of HIV-1 infection or in the pathogenesis of AIDS. We have isolated two biologically active HIV-1 clones: LW 12.3, which contains defects in the vif and vpr genes, and MN ST.1, which has a defect in the vpu gene. LW 12.3 is unable to replicate in peripheral blood mononuclear cells (PBMC). The growth of MN-ST.1 in SupT1 cells is marked by a 3-week lag in extracellular virus production and by the presence of unusually abundant viral buds. We demonstrate here that coinfection of PBMC with these two partially defective HIV-1 clones extends the cellular host range of LW 12.3, significantly increases the replication rate of both viral genomes, and eliminates the delay in production observed with the vpu-defective MN ST.1. When the lesions in vpr and vif of LW 12.3 are repaired, the resultant virus grows normally in PBMC. This is also the case when only vif is repaired, indicating that complementation of LW 12.3 in PBMC by MN ST.1 is mediated by vif in trans. The reciprocal complementation results in a dramatic increase of HIV-1 virulence. This two-component model represents a simplified version of the in vivo situation and illustrates one way in which interaction of defective viruses could increase the spread of infection and progression of disease.

Studies on serum-free culture of insect cells for virus propagation and recombinant protein production

Three lepidopteran cell lines, Spodoptera frugiperda cell line IPLB-Sf-21 and its clonal isolate Sf9 and the Trichoplusia ni cell line TN368, were adapted to a serum-free medium, EX-CELL 400. The cell growth kinetics of the serum-free cultures in monolayer were compared with that of serum-containing cultures. Virus production and recombinant protein production with a baculovirus expression vector in serum-free cultures were evaluated in comparison with those in serum-containing cultures. The production of both polyhedra and extracellular virus in both types of media was not significantly different. The production of β-galactosidase as a recombinant protein in the serum-free cultures was not as high as that in serum-containing cultures. Electron microscopical studies showed that virus replication and polyhedra formation in serum-free cultures were very similar to those in serum-containing cultures. The larval infectivity of polyhedra produced in the three cell lines grown in EX-CELL 400 medium did not differ from each other. In contrast, the infectivity of polyhedra produced in serum-containing cultures differed for the three cell lines and polyhedra from IPLB-SF-21 cells showed the highest infectivity.

Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.