Wild-type Infected Cells Research Articles

Leishmania pifanoi pathogenesis: selective lack of a local cutaneous response in the absence of circulating antibody.

Recently, a role for B cells in the pathogenesis associated with infection by Leishmania (Leishmania mexicana complex and L. donovani) has been established. In the case of L. mexicana complex parasites (L. mexicana, L. pifanoi, and L. amazonensis), a critical role for immunoglobulin G-mediated mechanisms for the amastigote stage in the host is evident; however, the immunological mechanisms involved remain to be established. In vitro analysis of the kinetics of parasite uptake by macrophages failed to indicate a major effect of antibody opsonization. Given the importance of CD4(+) T cells in the development of disease caused by these parasites, the possibility that the lack of pathogenesis was due to the lack of development of an immune response at the local site (draining lymph node and/or cutaneous site) was explored. Interestingly, the level of CD4(+)-T-cell activation (proliferation and cytokine) in draining lymph nodes from mice lacking circulating antibody (resistant) was found to be comparable to that in nodes from wild-type mice (susceptible) at 2, 5, and 10 weeks postinfection. However, antibody-deficient animals had markedly reduced numbers of monocytes and lymphocytes recruited or retained at the site of cutaneous infection in comparison to wild-type mice, indicating a selective impairment in the local cutaneous immune response. In vitro antigen presentation studies employing tissue-derived (opsonized) amastigotes demonstrated that L. pifanoi-infected FcR(-/-) macrophages, in contrast to comparably infected wild-type cells, failed to activate Leishmania antigen-specific T lymphocytes. These data, taken together, suggest that one possible mechanism for the role of antibody in pathogenesis may be to mediate parasite uptake and regulate the immune response at the local cutaneous site of infection.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.

Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.

Versatility of the accessory C proteins of Sendai virus: contribution to virus assembly as an additional role.

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.

SV40 Small T Antigen Enhances Progression to >G2 during Lytic Infection

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27kip1 were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins.

Tau-mediated process outgrowth is differentially altered by the expression of MAP2b and MAP2c in Sf9 cells.

It is well documented that the MAPs, MAP2 and tau, play pivotal roles in neurite outgrowth. Several isoforms of MAP2 and tau are coexpressed in neurons, suggesting that the pattern of neurite outgrowth results from a functional equilibrium among these isoforms. In the present study, by coexpressing two of these MAPs at the same time in Sf9 cells, we demonstrated that tau-mediated process outgrowth is affected differently by MAP2b and MAP2c. MAP2b impairs tau ability to induce process outgrowth. Tau affects MAP2c capacity to induce the formation of multiple processes. There is evidence that actin microfilaments (F-actin) are involved in the elaboration of tau-mediated process outgrowth in Sf9 cells. We compared the effects of MAP2b and MAP2c with the effects of tau on F-actin distribution and stability in Sf9 cells. In MAP2b- and MAP2c-expressing cells with processes, F-actin was redistributed. However, in MAP2b-expressing cells without processes, the distribution of F-actin appears to be similar to the one in wild-type infected cells. Collectively, these results indicate that MAP2b could impair the ability of MAP2c and tau to redistribute F-actin in Sf9 cells, thereby decreasing their capacity to induce process formation. Furthermore, MAP2b and MAP2c patterns of process outgrowth were differentially modified by depolymerization of F-actin by cytochalasin D (CD). As previously reported for tau-expressing cells, the MAP2b-expressing cells developed a higher number of processes per cell and a higher number of cells presented processes in the presence of CD. However, the number of cells with multiple processes was lower in MAP2b-expressing cells than in tau-expressing cells treated with CD at 24 h postinfection. This suggests that MAP2b exerts an effect on F-actin stability at an earlier stage of infection than tau. MAP2c had also some stabilizing effects on F-actin at an early stage of infection, since the percentage of cells presenting one process was similar to the nontreated cells. Therefore, MAP2b seems to have less capacity than MAP2c to redistribute F-actin but, nonetheless, both of these MAP2 isoforms exert a stabilizing effect on F-actin at an early stage of infection. Finally, by modifying phosphorylation we showed that MAP2c capacity to induce multiple processes is related to protein phosphorylation in Sf9 cells. Therefore, the differential effect of MAP2c and MAP2b on process outgrowth seems also to depend on protein phosphorylation.

The role of interferon in influenza virus tissue tropism.

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.

The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA.

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

Establishment and Characterisation of a Porcine Rubulavirus (LPMV) Persistent Infection in Porcine Kidney Cells

Porcine rubulavirus (LPMV) can establish persistent infections in porcine kidney cells. Cell cultures characterised at passages 25 and 65 demonstrated haemadsorption, formation of syncytia, and a slower growth rate. The nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) protein were present in all cells, although not to the same extent as in wild type infected cells. Incubation of the cell cultures with virus neutralising antibodies could not cure them from the infection. The cells were resistant to LPMV high multiplicity superinfection, but lysed rapidly upon infection with VSV. These cells thus fulfilled the criteria of a true persistent infection. Viral particles were released into the medium from the persistently infected cells as measured by HA and infection of PK-15 cells with medium from the persistently infected cells. The infectious titer of the virus released from the persistently infected cells was 3 logs lower compared to wild type virus, the HN titer still being comparable. Virus released from the persistently infected cells was unable to cause a lytic infection in PK-15 cells, and showed a reduced ability to spread when compared to a LPMV lytic infection.

The UL13 Protein Kinase and the Infected Cell Type Are Determinants of Posttranslational Modification of ICP0

The herpes simplex virus infected-cell protein 0 (ICP0) acts as a promiscuous transactivator of genes introduced into eukaryotic cells by transfection or infection. The protein is highly posttranslationally modified by phosphorylation and nucleotidylylation. We have examined the electrophoretic mobility and phosphorylation of ICP0 in Vero and rabbit skin cells infected with wild-type virus or viruses from which the UL13 gene (ΔUL13) encoding a protein kinase or the α22/US1.5 genes (Δα22/ΔUS1.5) encoding putative transcriptional factors has been deleted. We report the following: (i) The accumulation of ICP0 and the electrophoretic mobility of ICP0 were dependent on the nature of the infected cell type and the presence of UL13. ICP0 encoded by wild-type virus accumulated to maximum levels earlier in infected Vero cells and its electrophoretic mobility was slower than that made in rabbit skin cells. In both Vero and rabbit skin cells infected with the ΔUL13 virus, the prevailing ICP0 form migrated faster than that accumulating in the corresponding cells infected with wild-type virus. (ii) The alteration in electrophoretic mobility of ICP0 made in cells infected with ΔUL13 virus was due to the absence of the UL13 protein and not to failure of posttranslational modification of Δα22/ΔUS1.5 proteins inasmuch as the mobility of ICP0 in cells infected with Δα22/ΔUS1.5 virus could not be differentiated from that of wild-type infected cells. (iii) ICP0 is extensively phosphorylated in infected cells even in the absence of UL13 protein. ICP0 is, however, a substrate for the UL13 kinase inasmuch as ICP0 was phosphorylated in mixtures of immune complexes of ICP0 and UL13. Complexes containing ICP0 only or infected cell lysate proteins reacting with preimmune serum from the rabbit immunized with UL13 protein failed to phosphorylate ICP0. (iv) In the absence of UL13, ICP22 is overproduced—an imbalance attributed to UL13. Thus, ICP22 regulates both the utilization of splice acceptor sites and the longevity of ICP0 mRNA (K. L. Carter and B. Roizman, 1996,Proc. Natl. Acad. Sci. USA93, 12535–12540); UL13 is involved in the posttranslational modification of ICP0 and is required for both posttranslational processing and control of abundance of ICP22.

The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase.

In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with gamma(1)z34.5- mutants. The carboxyl-terminal 64 aa of gamma(1)34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1alpha in yeast, and both MyD116 and gamma(1)34.5 interact with protein phosphatase 1alpha in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the alpha subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2alpha-P phosphatase activity of gamma(1)34.5- virus infected cells is lower than that of mock-infected cells. The eIF-2alpha-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2alpha-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [32P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, gamma(1)34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2alpha to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.

An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking.

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.

Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity.

Four different insect cell lines that can be used as hosts for baculovirus infection were assayed for the presence of endogenous exoglycosidases. All four cell lines, derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, or Malacosoma disstria, contained N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, and sialidase activities. Exoglycosidase activities were found in cell lysates as well as cell-free supernatants from uninfected and wild-type baculovirus infected cells. Oligosaccharide analysis of cellular glycoproteins using lectins recognizing Gal beta 1, 3GalNAc, Gal beta 1, 4GlcNAc, and NeuAc alpha 2,6Gal demonstrated that only Gal beta 1,3GalNAc was present. The demonstration that these cells contain exoglycosidases raises the possibility that the oligosaccharides of baculovirus-expressed glycoproteins are subject to enzymatic degradation.

An HSV LAT Null Mutant Reactivates Slowly from Latent Infection and Makes Small Plaques on CV-1 Monolayers

A Herpes simplex virus type I (HSV-I) strain 17 mutant deleted between the Not l and Hpal restriction sites of the latency associated transcript (LAT) region has been constructed. The mutant, therefore, contains a deletion of the putative LAT promoter and is called 17N/H. The 17N/H isolate established latent infections in mice nearly as efficiently as its wildtype parent. However, like other LAT null mutants, 17N/H reactivates from explanted ganglia with much slower kinetics than its LAT competent parent. In tissue culture, although 17N/H produces as much virus per cell as its strain 17 parent, it produces small plaques. The small plaque phenotype appears to be due to the inability of the virus to be released from the infected cell into the medium, following low but not high multiplicities of infection (m.o.i.). The mutant was also shown to produce an aberrant LAT homologous transcript of 1.1 kb as well as overproduce an approximately 29,000-Da HSV-specific polypeptide, which is barely detectable in wildtype infected cells. Rescuants of the 17N/H defect were constructed using a 10-kb restriction fragment containing viral sequences spanning the deletion, make large plaques, and have reactivation patterns and infected cell gene product profiles indistinguishable from the 17 parent. This shows that the phenotypes observed in 17N/H are reversed when the deletion, or at most sequences within 5 kb of each side of the deletion, is corrected. The possibilities that the defect in vital egress from infected cell, the small LAT homologous transcript, and the accumulation of the 29,000 Da polypeptide are related to the delayed reactivation kinetics are discussed.

Defect in Entry and Altered Pathogenicity of a Polyoma Virus Mutant Blocked in VP2 Myristylation

We have examined effects of mutations of the minor capsid proteins VP2 and VP3 of polyoma virus. Infection of cells by mutants blocked in expression of either VP2 or VP3 by alterations of their initiator methionine codons led to the rapid selection of wild-type revertants. This reversion suggests a strong, if not absolute, requirement for both proteins in virus growth. A mutant virus, VP2*, in which alanine was substituted for glycine-2, expressed a nonmyristylated form of VP2 that was incorporated into virions. Studies of VP2* revealed a 15- to 20-fold lower specific infectivity and a delay in growth in both primary and established mouse cells compared to wild-type virus. Analysis of the growth delay indicated a defect in an early step of infection, at or prior to uncoating. Synthesis of viral DNA and VP1 was delayed by about 9 hr in mutant compared to that in wild-type infected cells. No evidence was obtained for an effect on either virus assembly (encapsidation) or stability of virions. Infection of mice by the VP2* mutant virus resulted in attenuated virus replication and tumor induction which were much more severely affected following intranasal inoculation than intraperitoneal inoculation.

Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four is mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40° with any of the four is mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.

Expression of Human Recombinant CD23 in Insect Cells

Human CD23 (low affinity receptor for IgE) has been expressed in insect cells (Sf9) using the baculovirus expression system and the baculovirus transfer vector pAc373. Insect cells infected with a recombinant baculovirus coding for CD23 synthesized a polypeptide not found in wild-type infected insect cells that had antigenic properties similar to natural CD23 produced in RPMI 8866 cells. Surface expression of recombinant CD23 was demonstrated by its ability to bind IgE. Recombinant CD23 expressed in insect cells had a slightly lower molecular weight (43 kDa) than that of natural CD23 (45 kDa) from RPMI 8866 cells as detected by SDS-PAGE followed by Western-blotting. Affinity-purified recombinant CD23 from infected insect cells showed B-cell growth promoting activitiy. These observations demonstrate for the first time that biologically active recombinant CD23 can be produced by the baculovirus expression system, thus providing a useful source of recombinant material to elucidate the biological functions of CD23.

Effect of polylysine on the early stages of infection of wild type pseudorabies virus and of mutants defective in gIII

The main pathway of adsorption of pseudorabies virus (PrV) to its host cells is via interactions between viral glycoprotein gIII and a cellular heparin-like receptor. Mutants of PrV deficient in glycoprotein gill adsorb by an alternative, slower pathway. Penetration into the cells of gIll − mutants is also delayed compared to penetration of wild type virus. We show here that polylysine enhances the adsorption of gill- mutants. Furthermore, in the presence of polylysine the adsorption of wild type virus involving the interactions of viral glycoprotein gill and the heparin-like cellular receptor is efficiently bypassed. Polylysine appears to promote virus adsorption by bridging the cellular and viral membranes. Polylysine not only stimulates adsorption of gIII − mutants but also promotes their internalization; the delay in the initiation of viral protein synthesis that is observed in cells infected with gIII − mutants compared to wild type infected cells is abrogated. Because it is unlikely that polylysine can substitute for two different functions of gIII, adsorption and penetration, the delay in the initiation of the infectious cycle in gIII −-infected cells is probably related to the defect in adsorption. Furthermore, polylysine can completely overcome the inhibitory effects of antisera against gIII, but not the inhibitory effects of antisera that affect a later stage of infection. It is unlikely therefore that polylysine can promote penetration directly and that gIII is involved directly in penetration. These results, as well as those obtained previously, show that while gill is essential for the efficient adsorption of PrV, it affects virus penetration only indirectly.

Herpes simplex virus-encoded ribonucleotide reductase: Evidence for the dissociation/ reassociation of the holoenzyme

35S-labeled cells infected with herpes simplex virus type 1 (HSV-1), temperature-sensitive (ts) mutant ts 1222 were used as a source of the large subunit of the viral ribonucleotide reductase (RR) to investigate the binding of the large (RR1) and small (RR2) subunits in the active enzyme. Mixing 35S-labeled RR1 from ts 1222 with unlabeled RR1/RR2 complex from wild type (wt) infected cells resulted in the formation of a complex between 35S-labeled RR1 and unlabeled RR2, indicating that the complex between the RR1 and RR2 subunits is dynamic and subunit dissociation/reassociation occurs during enzyme function. Similar results were obtained when unlabeled HSV-2 RR was substituted for HSV-1 RR, demonstrating that the holoenzyme can be formed the large subunit of HSV-1 RR and the small subunit of HSV-2.

Phosphorylation of middle T by pp60 c- src: A switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis

Substitution of phenylalanine for tyrosine 315 of the polyoma virus middle T (mT) protein lowers the incidence and limits the spectrum of tumors induced following inoculation of the virus into newborn mice. This substitution removes the major site of phosphorylation by pp60 c- src without altering the ability of mT to associate with or to activate pp60 c- src . The mutant mT fails to show binding of a phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity that is normally present in wild-type mT complexes. Furthermore, an anti-peptide antiserum that specifically recognizes mT lacking phosphate at tyrosine 315 precipitates binary (mT-pp60 c- src ) but not ternary (mT-pp60 c- src -PtdIns 3-kinase) complexes from wild-type infected cell extracts. Reprecipitation with either anti-pp60 c- src or anti-mT serum brings down ternary complexes containing mT phosphorylated on tyrosine 315. Phosphorylation of mT by pp60 c- src in vivo is therefore a critical event for binding of PtdIns 3-kinase and for expression of the full tumorigenic potential of the virus.