Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene.

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

The delayed early gene G23 of temperate mycobacteriophage L1 regulates the expression of deoxyribonuclease, the product of another delayed early gene of the phage.

To get clues about the genes as well as the gene regulatory circuit controlling the lytic development of temperate mycobacteriophage L1, previously we screened several conditional lethal mutants of L1 and characterized some of them to an extent. One of the mutants, L1 G23ts23, was found defective in both growth and late gene transcription at 42 degrees C but not at 32 degrees C. Here we show that the above phage mutant is also defective in the expression of phage-coded deoxyribonuclease (DNase) at 42 degrees C but not at 32 degrees C. The G23 gene however does not code for the above enzyme. Further analyses using the L1 G23ts23 mutant suggest that synthesis of DNase is also not regulated by G23 at transcriptional level. Expression of functional DNase in fact requires de novo protein synthesis. Among the 25 revertants isolated from the L1 G23ts23 mutant, which are capable of growing at 42 degrees C (by overcoming the ts defect in late transcription), two, R4 and R22, have been shown to retain the ts defect in the expression of the above enzyme and R4, to retain also the G23ts23 mutation. This suggests that R4 (R22 was not tested for the presence of G23ts23 mutation) carries an extragenic suppressor of G23ts23 mutation in a different gene (we call this putative gene as Gx), which now helps bypass the requirement of G23 for late gene transcription. Possible role of G23 on the regulation of L1-coded Gx and deoxyribonuclease has been discussed at length.

Tobacco mosaic virus defective RNAs expressing C-terminal methyltransferase domain sequences are severely impaired in long-distance movement in Nicotiana benthamiana.

Tobamovirus replicase proteins, which function in replication and gene expression, are also implicated in viral cell-to-cell and long-distance movement. The role(s) of Tobacco mosaic virus (TMV) 126-/183-kDa replicase protein in the complex movement process are not understood due to lack of systems that can separate the multiple steps involved. We previously developed a bipartite TMV-defective RNA (dRNA) system to dissect the role of the N-terminal methyltransferase (MT) domain in accumulation and cell-to-cell movement of dRNAs [Knapp, E., Danyluk, G.M., Achor, D., Lewandowski, D.J., 2005. A bipartite Tobacco mosaic virus-defective RNA (dRNA) system to study the role of the N-terminal methyltransferase domain in cell-to-cell movement of dRNAs. Virology 341, 47-58]. In the current study we analyzed long-distance movement of dRNAs in the presence of helper virus in Nicotiana benthamiana. dRNAs expressing approximately 50% of the MT domain (DeltaHinc151) moved long-distances in more than half of the plants. dRNAs expressing approximately 90% of the MT domain sequences (DeltaCla151) predominantly failed to accumulate in upper leaves. The helper virus moved systemically when inoculated alone or with a dRNA. In inoculated leaves, more DeltaHinc151-induced infection foci spread adjacent to class V veins compared to those of DeltaCla151. Consequently, DeltaHinc151 infected more class V veins than DeltaCla151. DeltaCla151 was only detected in bundle sheath cells, whereas DeltaHinc151 could accumulate in bundle sheath and phloem parenchyma cells of class V veins. However, the latter accumulation pattern did not always result in systemic accumulation of DeltaHinc151, suggesting that factors in addition to those affecting cell-to-cell movement played a role in long-distance movement.

Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth.

There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin::angiostatin or endostatin::kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment.

A bipartite Tobacco mosaic virus-defective RNA (dRNA) system to study the role of the N-terminal methyl transferase domain in cell-to-cell movement of dRNAs.

Plant viruses, in particular Tobacco mosaic virus (TMV), are model systems to study RNA and protein trafficking in plants. Although TMV cell-to-cell transport controlled by the 30-kDa movement protein (MP) has been intensively studied, it was only recently demonstrated that the 126/183-kDa replicase proteins are also involved in cell-to-cell movement. Elucidating the role(s) of 126/183-kDa proteins in movement is complicated because these proteins have multiple functions associated with replication and gene expression. To overcome these difficulties we developed a TMV helper virus-defective RNA (dRNA) system to study the role of replicase protein sequences in dRNA cell-to-cell movement. Artificially constructed dRNAs lacking sequences encoding the helicase and polymerase domains of the replicase proteins and portions of the MP were viable in protoplasts and plants in the presence of helper virus. Expression of at least approximately 50% of the methyl transferase (MT) domain was required for efficient dRNA movement in Nicotiana benthamiana. dRNAs that encoded the N-terminal 64 replicase amino acids or lacked a translatable MT domain failed to move or moved poorly. TMV dRNAs expressing 258 amino acids of the replicase protein moved into all specialized non-vascular tissues, whereas dRNAs expressing replicase sequences beyond amino acid 258 were restricted to the epidermis and palisade mesophyll tissues. Furthermore, second-site mutations within the dRNA-encoded truncated replicase protein altered efficiency in dRNA cell-to-cell movement.

Development of HSV-specific CD4+ Th1 responses and CD8+ cytotoxic T lymphocytes with antiviral activity by vaccination with the HSV-2 mutant ICP10DeltaPK.

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10DeltaPK) protects from HSV-2 challenge in the mouse and guinea pig cutaneous and vaginal models and reduces the incidence and frequency of recurrent disease (Vaccine (17) (1999) 1951; Vaccine (19) (2001) 1879). The present studies were designed to identify the immune responses induced by ICP10DeltaPK and define the component responsible for protective activity. We found that ICP10DeltaPK elicits a predominant HSV-specific T helper type 1 (Th1) response, as evidenced by: (1) higher levels of HSV-specific IgG2a (Th1) than IgG1 (Th2) isotypes and (2) higher numbers of CD4+ IFN-gamma than IL-10 secreting T cells in popliteal lymph nodes. This Th1 response pattern was associated with a significant increase in the levels of IL-12 produced by dendritic cells from ICP10DeltaPK than HSV-2 immunized animals. Lymph node cells (LNCs) from ICP10DeltaPK immunized mice had significantly higher levels of HSV-2 specific cytolytic activity than LNCs from mice immunized with HSV-2 and it was mediated by CD8+ T cells. CD8+ CTL were not seen in LNCs from HSV-2 immunized mice. In adoptive transfer experiments, CD8+ T cells and, to a lower extent, CD4+ T cells from ICP10DeltaPK immunized mice inhibited HSV-2 replication, suggesting that they are involved in the protective immunity induced by ICP10DeltaPK vaccination.

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.

A growth and latency compromised herpes simplex virus type 2 mutant (ICP10DeltaPK) has prophylactic and therapeutic protective activity in guinea pigs.

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10DeltaPK) protects from fatal HSV-2 challenge in the mouse model (Aurelian L, Kokuba H, Smith CC. Vaccine potential of a Herpes Simplex Virus type 2 mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10). Vaccine 1999;17:1951-1963). Here we report the results of our studies with ICP10DeltaPK in the guinea pig model of recurrent HSV-2 disease. ICP10DeltaPK was also compromised for growth and disease causation in this model. It was not isolated from latently infected ganglia by explant co-cultivation. The proportions of latently infected ganglia were significantly lower for ICP10DeltaPK than HSV-2 [3/25 (12%) and 7/10 (70%), respectively]. Similar results were obtained for the levels of viral DNA (8 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and HSV-2, respectively]. ICP10DeltaPK immunization caused a significant (P< or = 0.001) decrease in the proportion of animals with primary [1/14 (6%) and 16/16 (100%) for ICP10DeltaPK and PBS, respectively) and recurrent [1/14 (6%) and 11/14 (79%) for ICP10DeltaPK and PBS, respectively) HSV-2 skin lesions. It also protected from genital HSV-2 disease [1/10 and 10/10 for ICP10DeltaPK and PBS, respectively] and decreased the severity of the lesions in both models. Quantitative PCR (Q-PCR) with primers that distinguish between HSV-2 and ICP10DeltaPK indicated that immunization reduced the proportion of ganglia positive for HSV-2 DNA [8/25 (32%) and 7/10 (70%) for ICP10DeltaPK and PBS, respectively) and its levels [3 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and PBS, respectively]. The proportion of HSV-2 infected animals with recurrent disease was also significantly (P < or = 0.001) decreased by immunization with ICP10DeltaPK [1/15 (7%) and 11/14 (79%) with recurrent disease for ICP10DeltaPK and PBS, respectively], suggesting that ICP10DeltaPK has prophylactic and therapeutic activity in the guinea pig.

Retention of a small replicase gene segment in tomato bushy stunt virus defective RNAs inhibits their helper-mediated trans-accumulation.

Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.

High-level expression of active HIV-1 integrase from a synthetic gene in human cells.

A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.

Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins.

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.

The exonuclease activity of HSV-1 UL12 is required for in vivo function.

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear localization, suggesting that they have retained an intact global conformation. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determined whether expression of mutant proteins from amplicon plasmids could complement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so. Loss of exonuclease activity therefore correlates with loss of in vivo function.

Mechanisms of replication-deficient vaccinia virus/T7 RNA polymerase hybrid expression: effect of T7 RNA polymerase levels and alpha-amanitin.

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).

Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro.

We analyzed the ability of a recombinant replication-defective adenovirus vector with the carcinoembryonic antigen (CEA) promotor to transfer the thymidine kinase gene of herpes simplex virus (HSVtk) into gastric cancer cells to confer sensitivity to ganciclovir (GCV). CEA-producing gastric cancer cell lines (MKN28 and MKN45), a CEA-nonproducing gastric cancer cell line (MKN1), and a human uterine cervical cancer cell line (HeLa) were infected with a recombinant adenovirus carrying lacZ reporter gene coupled to the CEA promoter (AdCEAlacZ). The efficiency of AdCEAlacZ-mediated gene transfer was correlated with the amount of CEA produced by each cell line. Furthermore, the 50% growth inhibitory concentrations (IC50) of GCV were 21 and 5.8 microm for MKN28 and MKN45, respectively, when infected with a recombinant adenovirus carrying the HSVtk gene coupled to the CEA promoter (AdCEAtk). However, MKN1 and HeLa cells infected with AdCEAtk remained resistant to GCV (IC50 > 300 microm of GCV). In addition, a bystander killing effect was demonstrated against MKN45 cells when only 20% of AdCEAtk-infected cells were mixed with uninfected cells. These data indicate the potential for targeted gene therapy using the cell type-specific promotor of the CEA gene against gastric cancers that produce CEA.

Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging.

To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells. Following infection of breast cancer cells with an adenovirus expressing beta-galactosidase gene, high levels of beta-galactoside activity were observed. No beta-galactosidase activity was observed in low-density human bone marrow cells. A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing beta-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency. In contrast, human bone marrow cells treated under the same conditions expressed very low levels of transfected beta-galactosidase DNA. Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments. A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells. Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells. However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine. On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.

Reactivation of thymidine kinase-defective herpes simplex virus is enhanced by nucleoside.

Herpes simplex virus (HSV) mutants defective for thymidine kinase expression (TK-) have been reported to establish latent infection of sensory ganglia of mice, in that HSV latency-associated transcript is expressed, but to be defective for reactivation. In the present study, the mechanism of defective reactivation by TK- HSV was investigated. Latent infection established by each of three reactivation-defective HSV type 1 mutants was studied. Reactivation in explant culture was markedly enhanced by the addition of thymidine (dTdR) to the explant culture medium. Without added dTdR, reactivation occurred in 0 of 32 ganglia, while when dTdR (200 microM) was present, reactivation occurred in 32 of 37 ganglia (86%). Reactivation was minimal or did not occur after treatment with other nucleosides; specificity for dTdR would suggest the importance of dTdR nucleotide levels rather than more general nucleotide pool imbalance. Enhanced reactivation by dTdR was dose dependent and was blocked by acyclovir. While some degree of inhibition of TK- HSV by acyclovir may be expected, the complete block of dTdR-enhanced reactivation was unexpected. This result may suggest that HSV is particularly vulnerable during initial reactivation events. The mechanism of dTdR-enhanced reactivation of TK- HSV was further evaluated during in vivo infection by TK- HSV. For mice infected with TK- HSV, virus was undetectable in ganglia 3 days later. However, for mice infected with TK- HSV and treated with dTdR, virus was readily detected (2.8 x 10(3) PFU per ganglion). This result suggested that in vivo treatment with dTdR enhanced replication of TK- HSV in ganglion neurons. In turn, this suggests that in latently infected ganglia, dTdR-enhanced reactivation of TK- HSV occurred as a result of viral replication in neurons following initial reactivation events.

Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) stimulates viral infectivity. The mechanism of this phenotype was investigated. Viruses containing disrupted nef genes were 4 to 40 times less infectious than wild-type HIV-1 in a single-round infection. The Nef-mediated stimulation HIV-1 infectivity was dependent on the association of Nef with the plasma membrane and could be observed when Nef was provided in trans in the virus producer but not target cells. The impaired infectiousness of nef-defective (delta Nef) virions was observed whether or not CD4 was present in either of these cells. Furthermore, it was independent of the mode of viral entry, since it was not rescued by pseudotyping Env- HIV-1 virions with the amphotropic murine leukemia virus envelope glycoproteins. As predicted from this result, wild-type and delta Nef virions entered cells with equal efficiencies. However, despite their normal content in viral genomic RNA and reverse transcriptase activity, delta Nef viruses were limited in their ability to perform reverse transcription once internalized in several cell types, including peripheral blood lymphocytes. Since Nef does not appear to be abundant in virions, these results suggest that Nef acts in producer cells to allow the generation of particles fully competent for completing steps that follow entry, leading to efficient reverse transcription of the HIV-1 genome. Using a trans complementation assay, we found that Nef proteins from a number of primary HIV-1 isolates as well as, to a milder degree, those from HIV-2ST and SIVMAC239 could enhance the infectivity of delta Nef HIV-1. This indicates that the Nef-mediated stimulation of proviral DNA synthesis is highly conserved and likely plays an important role in vivo.

Analysis of human immunodeficiency virus type 1 integrase mutants.

The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication.

Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX.

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.