Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

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.     

Recruitment of cellular recombination and repair proteins to sites of herpes simplex virus type 1 DNA replication is dependent on the composition of viral proteins within prereplicative sites and correlates with the induction of the DNA damage response

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.     

Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.     

The Kinase Activity of Ataxia-Telangiectasia Mutated Interferes with Adenovirus E4 Mutant DNA Replication

Adenovirus (Ad) mutants that lack early region 4 (E4) are unable to produce the early regulatory proteins that normally inactivate the Mre11/Rad50/Nbs1 (MRN) sensor complex, which is a critical component for the ability of cells to respond to DNA damage. E4 mutant infection therefore activates a DNA damage response, which in turn interferes with a productive viral infection. MRN complex proteins localize to viral DNA replication centers in E4 mutant-infected cells, and this complex is critical for activating the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which phosphorylate numerous substrates important for DNA repair, cell cycle checkpoint activation, and apoptosis. E4 mutant growth defects are substantially rescued in cells lacking an intact MRN complex. We have assessed the role of the downstream ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. We did not identify a role for either ATM or ATR in “repair” of E4 mutant genomes to form concatemers. ATR was also not observed to contribute to E4 mutant defects in late protein production. In contrast, the kinase activity of ATM was important for preventing efficient E4 mutant DNA replication and late gene expression. Our results suggest that the MRN complex interferes with E4 mutant DNA replication at least in part through its ability to activate ATM.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.