Infection with Langat Flavivirus or expression of the envelope protein induces apoptotic cell death.

Langat (LGT) flavivirus, derived from infectious full-length cDNA clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (Neuro-2a) and simian kidney (Vero and LLC-MK(2)) cells. The hallmark of apoptosis, cleavage of cellular DNA, was observed 48 h after infection of Vero, LLC-MK(2), and Neuro-2a cells by electrophoresis analysis. Apoptosis in infected cells was also confirmed by TUNEL assay. LGT-infected Neuro-2a cells showed an increase in caspase-3-like protease (DEVDase) activity. Expression of the major envelope glycoprotein (E) alone reduced cell viability in both Vero and Neuro-2a cells, and the baculovirus P35 protein, which inhibits multiple caspases, completely blocked this effect. Cleavage of cellular DNA was observed in E gene-transfected Vero cells by TUNEL assay. Expression of E protein or caspase-9 resulted in activation of caspase-3-like proteases in Neuro-2a cells. The caspase-3-like protease specific inhibitor, Ac-DEVD-CHO peptide, partially inhibited E protein- or caspase-9-induced apoptosis in Neuro-2a cells. These observations indicate that infection of cells with LGT virus or expression of LGT virus E protein induces apoptosis through a caspase-3-like protease pathway.

The baculovirus PE38 protein augments apoptosis induced by transactivator IE1.

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.

In vivo and in vitro analysis of baculovirus ie-2 mutants.

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.

Role of baculovirus IE2 and its RING finger in cell cycle arrest.

The ie2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is known to transactivate transient expression from viral promoters in a host cell-specific manner. We report that transfection of Spodoptera frugiperda (SF-21) cells with ie2 was sufficient to arrest the cell cycle, resulting in the accumulation of enlarged cells with abnormally high DNA contents. By 72 h posttransfection, more than 50% of ie2-transfected cells had DNA contents greater than 4N. There was no evidence of mitotic spindle formation in these cells, and expression of ie2 appeared to block cell cycle progression in S phase. Several ie2 mutants were analyzed to further define the region of IE2 responsible for arresting the cell cycle. Analysis of these mutants showed that deletion of the RING finger motif eliminated the ability of IE2 to arrest the cell cycle but did not affect its ability to transactivate the ie1 promoter. Moreover, mutation of a single conserved cysteine (C251) of the RING finger motif abolished the ability of IE2 to block cell cycle progression but had no apparent effect on its transregulatory activity. In contrast, a mutant of IE2 containing a deletion of residues 94 to 173 was able to block cell division but lacked trans-regulatory activity. Thus, the ability of IE2 to arrest the cell cycle depended on the integrity of the RING finger motif and was distinct from and independent of its ability to trans-activate the ie1 promoter. IE2 also arrested the division of cells derived from other insect species, Trichoplusia ni (TN-368 and BTI-TN-5B1-4) and Helicoverpa zea (Hz-AM1).

Induction of apoptosis by baculovirus transactivator IE1.

Apoptosis is induced upon infection of SF-21 cells by mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) lacking a functional p35 gene which encodes a stoichiometric inhibitor of members of the interleukin-1beta converting enzyme family of cysteine proteases (N.J. Bump et al, Science 269:1885-1888, 1995; R.J. Clem, M. Fechheimer, and L.K. Miller, Science 254:1388-1390, 1991). We found that transfection of SF-21 cells with the AcMNPV ie-1 gene was sufficient to induce apoptosis, which was characterized by fragmentation of cellular DNA into oligonucleosomal fragments and apoptotic body formation. No signs of apoptosis were observed in Trichoplusia ni TN-368 cells transfected with ie-1, a result which is consistent with the observation that p35 mutants of AcMNPV do not induce apoptosis in this cell line. Cotransfection of SF-21 cells with p35 blocked ie-1-induced apoptosis, indicating that expression of ie-1 activates apoptosis through a p35-inhibitable cysteine protease pathway. Cotransfection with Cp-iap, an active member of another family of antiapoptotic inhibitors of apoptosis (iaps), also inhibited IE1-induced apoptosis. Thus, ie-1 may participate in inducing apoptosis in AcMNPV-infected cells, although the dependence of induction on DNA replication suggests that ie-1 is not the direct apoptotic signal during infection. The ie-1 gene product, IE1, is known to be a potent transactivator of baculovirus gene expression that interacts with specific palindromic sequences which can act as transcriptional enhancers and as origins of DNA replication in transient assays.

Biochemical characterization of VspI methyltransferase.

The gene (vspIM) encoding VspI methyltransferase (MTase) has previously been cloned and sequenced, and shown to belong to the gamma class of m6-adenine MTases [Degtyarev et al., Nucleic Acids Res. 21 (1993) 2015]. Here it is shown that the MTase modifies the third adenine within the recognition sequence 5'-ATTAAT-3'.

Interaction of VspI and Tru9I restriction endonucleases with synthetic oligonucleotides.

We describe the properties of two new restriction endonucleases VspI and Tru9I which recognize sequences AT TAAT and T TAA, respectively. The molecular weights, subunit structure and steady-state kinetic constants of these enzymes for native and modified substrates have been determined. We have investigated the interaction of VspI and Tru9I with synthetic oligonucleotides containing modifications either within the recognition sites or around them. These modifications represent the substitution of different DNA deoxyribonucleosides by 1,2-dideoxy-D-ribofuranose, which corresponds to loss of the heterocyclic base while the sugar-phosphate chain remains intact. The effects of the substitutions were analyzed by determining the steady-state kinetic values of the hydrolysis reaction by VspI and Tru9I. The enzymes exhibited Michaelis-Menten kinetics for hydrolyzable substrates. The initial rates (V0) of hydrolysis of modified and unmodified strands of the duplexes varied as a result of these substitutions. The substrates for VspI and Tru9I which contain modifications around the bond to be hydrolyzed or within the complementary nucleosides were unreactive.

[The structure of marriages and human resistance to the disease of alcoholism]. / Struktura brakov i ustoichivost' cheloveka k zabolevaniiu alkogolizmom.

Genealogies of men suffering from alcoholism (574) and healthy men (368) are investigated. It is revealed that persons from endogamous marriages are more frequently found among people suffering from alcoholism than among healthy persons (36.6% and 25.2%). 55% of men of exogamous origin have relatives suffering from alcoholism, 40% of men from endogamous marriages have such relatives. It is supposed that high heterozygosity decreases the risk of alcoholism.