Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.     

Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.     

Structural state of newly replicated closed circular simian virus 40 DNA.

We have studied the early transition of newly replicated, segregated daughter molecules of simian virus 40 (SV40) into their mature, fully supercoiled state. The DNA of SV40 replicating in African green monkey kidney CV1 cells was chronically labeled with [14C]thymidine and pulse-labeled with [3H]thymidine. The cells were lysed and the viral DNA was isolated. Density gradient centrifugation of viral DNA in cesium chloride revealed that the pulse-labeled, newly synthesized, closed circular supercoiled DNA molecules banded at a slightly higher density (delta sigma = 0.0025) than the chronically labeled DNA, suggesting that the newly completed molecules were in a different structural state. Electrophoresis of DNA in agarose gels at appropriate chloroquine concentrations demonstrated that the mobility of the pulse-labeled closed, superhelical DNA was retarded relative to that of the chronically labeled DNA. These observations indicated that the newly completed SV40 DNA molecules existed in a structural state more relaxed than that of mature DNA by one or two linking numbers.     

Adenovirus DNA is associated with the nuclear matrix of infected cells.

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.     

The HIV-1 passage from cytoplasm to nucleus: the process involving a complex exchange between the components of HIV-1 and cellular machinery to access nucleus and successful integration

The human immunodeficiency virus 1 (HIV-1) synthesizes its genomic DNA in cytoplasm as soon as it enters the cell. The newly synthesized DNA remains associated with viral/cellular proteins as a high molecular weight pre-integration complex (PIC), which precludes passive diffusion across intact nuclear membrane. However, HIV-1 successfully overcomes nuclear membrane barrier by actively delivering its DNA into nucleus with the help of host nuclear import machinery. Such ability allows HIV-1 to productively infect non-dividing cells as well as dividing cells at interphase. Further, HIV-1 nuclear import is also found important for the proper integration of viral DNA. Thus, nuclear import plays a crucial role in establishment of infection and disease progression. While several viral components, including matrix, viral protein R, integrase, capsid, and central DNA flap are implicated in HIV-1 nuclear import, their molecular mechanism remains poorly understood. In this review, we will elaborate the role of individual viral factors and some of current insights on their molecular mechanism(s) associated with HIV-1 nuclear import. In addition, we will discuss the importance of nuclear import for subsequent step of viral DNA integration. Hereby we aim to further our understanding on molecular mechanism of HIV-1 nuclear import and its potential usefulness for anti-HIV-1 strategies.     

A fixed site of DNA replication in eucaryotic cells

We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with ³H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.     

Reassociation kinetics of deoxyribonucleic acid in antigen-stimulated mouse-spleen cells.

In order to directly compare the complexity of the genome of lymphoid cells which have been antigenically stimulated, with that of non-immunized and non-lymphoid cells, DNA was pulse labeled and extracted from BALB/c mouse spleen cells at various time intervals after antigenic stimulation in vivo; the reassociation rates of these newly synthesized DNA preparations were compared with those of the total mouse spleen DNA, obtained from same sources and at the same times. DNA labeled for 60 min at 43, 53, or 72 h after antigenic restimulation, reassociated faster than the corresponding total DNA. On the other hand, the ressociation profile of DNA, labeled for 60 min during the first 24 after restimulation did not differ from that of the total DNA extracted at the same time. When labeled thymidine was available for incorporation at a constant concentration over a period of 24 h, reassociation patterns of labeled DNA were identical to those of the corresponding total DNA at all times after restimulation. Newly synthesized nuclear DNA exhibited reassociation profiles identical to those of the corresponding total nuclear DNA at all times tested. Also, no differences between the reassociation rates of nuclear and total cellular DNA were observed. It was concluded that antigenic stimualtion does not induce a major amplification of genes in the stimulated cells, and that the rapidly reassociating DNA species described represent extranuclear (cytoplasmic) DNA.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Caffeine-induced reorganization of DNA replicating system occurs on or near nuclear matrix in HeLa cells

Since caffeine reorganizes the DNA replicating system, with several consequences, we studied the effect of caffeine on the DNA replication which normally occurs on or near the nuclear matrix in a variety of eukaryotic cells. When HeLa cells, treated with or without the DNA-damaging agent, neocarzinostatin, were postincubated in the presence or absence of caffeine and then pulse-labeled with [3H]thymidine, the DNA remaining tightly associated with the matrix was enriched in the newly synthesized DNA at the same level as that seen in untreated cells. The nuclear matrix-bound DNA polymerase alpha activity was also the same in these cells. Therefore, in the presence of caffeine, DNA replication, with or without DNA damage, also occurs on or near the nuclear matrix, as is the case in normal DNA replication.     

Rifampin and vaccinia DNA.

The effect of rifampin on the replication of vaccinia DNA was studied in mouse L cells by a cytochemical techinque and by alkaline sucrose sedimentation analysis of newly synthesized viral DNA molecules. By the use of a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral "factories" in rifampin-treated, virus-infected cells were found to be indistinguishable from those observed in untreated, infected cells. Sedimentation analysis in alkaline scurose gradients of the viral DNA molecules labeled in pulse-chase experiments showed that formation of small fragments, elongation into "intermediate"-sized molecules, and maturation into full-length viral DNA and, finally, into cross-linked viral DNA molecules occurred in the absence or presence of rifampin. The results support the view that the primary effect of the drug is related to assembly or morphogenesis.     

Preferential association of newly synthesized histones with replicating SV40 DNA.

The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA.     

Hyperthermia blocks DNA processing at the nuclear matrix

The capacity of control and heated HeLa cells to process newly polymerized DNA at the nuclear matrix was measured. DNA which had been pulse-labeled with [3H]thymidine was enriched by a factor of up to 6 at the cell's nuclear matrix. During continuous exposure to [3H]thymidine at 37 degrees C this enrichment for pulse-labeled DNA was reversed with a half-time of 7 min. We interpret this processing of newly replicated DNA to be a distribution of newly polymerized DNA throughout replicon-sized nuclear DNA domains. Both processing of newly polymerized DNA at the nuclear matrix and ligation of replicon clusters into the interphase cell chromosome were halted by incubation of cells at temperatures at or above 43 degrees C. When HeLa cells were pulse-labeled during a 30-min incubation at 45 degrees C and replaced at 37 degrees C, the enrichment for 3H-labeled DNA at the nuclear matrix was reversed with an initial half-time of 4 h. The results indicate that exposure of cells to hyperthermic temperatures blocks ongoing nascent DNA processing at the nuclear matrix and results in a retardation of DNA processing in preheated cells replaced at 37 degrees C.     

SV40 virions and viral RNA metabolism are associated with cellular substructures

Nuclear matrices were prepared by DNase and high salt extraction of SV40-infected epithelial monkey cells. The matrices retain the majority of SV40 virions. This conclusion is based on electron microscopic observations of the occurrence of encapsidated viral DNA that is resistant to DNase digestion and on the analysis of viral proteins by gel electrophoresis. Pulse labeled SV40 RNA is also associated with the nuclear matrix (less than 15% of the viral RNA is removed by DNase and high salt). Pulse-chase experiments revealed that processing of SV40 RNA takes place on the nuclear matrix and the processed molecules are directly transported to the cytoplasm where they are associated with the cytoskeleton. These results suggest a central role for the nuclear and cytoplasmic substructures in virus assembly and in the biogenesis of viral RNA.     

The nuclear matrix continues DNA synthesis at in vivo replicational forks

Alkaline cesium chloride gradient analysis of in vivo [3H]bromodeoxyuridine-labeled and in vitro [alpha-32P]dCTP-labeled DNA was used to determine whether in vitro DNA synthesis in regenerating rat liver nuclei and nuclear matrices continued from sites of replication initiated in vivo. At least 70 and 50% of the products of total nuclear and matrix-bound in vitro DNA synthesis, respectively, were continuations of in vivo initiated replicational forks. The relationship of the in vitro DNA synthetic sites in total nuclei versus the nuclear matrix was examined by using [3H]bromodeoxyuridine triphosphate to density label in vitro synthesized DNA in isolated nuclei and [alpha-32P]dCTP to label DNA synthesized in isolated nuclear matrix. A minimum of about 40% of matrix-bound DNA synthesis continued from sites being used in vitro by isolated nuclei. Furthermore, nuclear matrices prepared from in vitro labeled nuclei were 5-fold enriched in DNA synthesized by the nuclei and were several-fold enriched, compared to total nuclear DNA, in a particularly high density labeled population of DNA molecules.