The 65K DNA binding protein appears early in HSV-1 replication

Summary The infectivity of influenza A viruses like fowl plague virus (FPV) with a cleaved hemagglutinin (HA) is highly sensitive to treatment at pH 5, while strains like PR 8 or virus N with a noncleaved HA survive under this condition. After double infection of chick embryo cells with FPV and PR 8 or virus N, the yield of virus with the HA gene of FPV is greatly reduced. However, it can now survive treatment at pH 5, and the surviving FPV particles form plaques only in the presence of trypsin, indicating that they were coated by the HA of PR 8 or virus N, depending on the coinfecting virus. The results are discussed with respect to the build-up and maintenance of a large reservoir of nonpathogenic influenza A viruses with noncleavable HA in water fowl.     

Residues F593 and E596 of HSV-1 tegument protein pUL36 (VP1/2) mediate binding of tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument proteins pUL36 (VP1/2) and pUL37 are essential for secondary envelopment during the egress of viral particles. Our laboratory has previously shown that HSV-1 pUL36(512-767) fragment interacts with full-length pUL37. A number of single and double amino acid changes of conserved residues in the pUL36(512-767) fragment were generated using alanine-scanning site-directed mutagenesis. The interaction of pUL36(512-767) and pUL37 was then assessed using a combination of yeast two-hybrid and coimmunoprecipitation assays. Single changes to alanine of pUL36 residues F593 and E596 impaired binding of pUL37 with the greatest effect observed for the substitution E596A. Double mutations involving either of these residues in combination with the substitution E580A essentially blocked binding of pUL37. This information will provide the basis for generation of viral mutants to further define the importance of the pUL36/pUL37 interaction in assembly of HSV-1.     

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.     

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.     

Evidence that SV40 VP1-DNA interactions contribute to the assembly of 40-nm spherical viral particles

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self-assembles into virus-like particles (VLPs) of approximately 40 nm in diameter when over-expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40-nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2,000 bp is packaged into the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.     

Evidence that virion-associated VP1 of avibirnaviruses contains viral RNA sequences

Summary.  The VP1 encoded by genomic segment B of birnaviruses is generally known to exist as a genome-linked protein (VPg) and as a “free” polypeptide of 90 kDa in virus particles. The guanylylation activity associated with infectious bursal disease virus (IBDV) was demonstrated by incubating purified virus in presence of [] GTP; optimum activity in the 90 kDa form of VP1 was seen in low salt concentration in the presence of 4 mM magnesium ions over a wide range of incubation temperatures. The IBDV VP1 was shown to lack guanyl transferase activity. Northwestern (RNA-protein) blot analysis of purified virus using a radiolabeled cDNA probe consisting of and ends of genomic segment B indicated that both forms of virion-associated VP1 contained viral RNA sequences of which those linked to VPg corresponded to the two genome segments and those linked to the 90 kDa VP1 were probably a short oligonucleotide of the terminal viral RNA sequences. Received August 23, 1996 Accepted October 29, 1996     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Host cell and virus strain differences in synthesis of immediate early polypeptides in HSV-1 infected cells

The synthesis of the immediate early (IE) polypeptides was analysed in primary rabbit kidney (RK) cells and a stable line of rabbit lung (ZP) cells infected with the syncytial (syn) strain HSZP and the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: After cycloheximide reversal the infection of RK and ZP cells with HSZP strain led to synthesis of five IE polypeptides (175K, 136K, 87K, 68K, and 63K), while infection of both cell cultures with the KOS strain led to synthesis of significantly reduced amounts of the IE polypeptides. The ability to switch on the expression of non-alpha viral genes was impaired in RK cells infected with the HSZP strain. The IE polypeptides were still detectable without any sign of the non-IE polypeptide synthesis 4 hr after cycloheximide reversal. The observed failure of the IE HSZP polypeptides to undergo posttranslational modification in ZP cells may be the consequence of this phenomenon. In contrast to the KOS IE mRNAs, the HSZP IE mRNAs exhibited a pronounced functional stability in both cell cultures. The IE polypeptides were still synthesized in HSZP-infected cells which had been incubated for 19 hr after cycloheximide reversal in the presence of actinomycin D (Act D). The HSZP strain failed to suppress the host polypeptide synthesis in RK but not in ZP cells. However, the HSZP strain, in contrast to the KOS strain, proved to be defective with respect to the early shutoff of host polypeptide synthesis in both cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus type 1 (HSV-1) HSZP interferes also heat inactivated with early shutoff of host protein synthesis induced by HSV-1 KOS

The ability of two strains of herpes simplex virus type 1 (HSZP and KOS) to shut off the host protein synthesis in the presence of Actinomycin D was investigated. The HSZP strain proved to be defective with respect to the so-called early shutoff function. In superinfection experiments, the HSZP was effective at interfering with the early shutoff function of the KOS strain provided that the HSZP infection preceded KOS superinfection. Heat inactivation of the HSZP did not lead to the loss of its interfering ability. Evidence was given that this interference was neither due to the hindrance of the KOS by HSZP at adsorption nor due its exclusion during penetration.     

Herpes simplex virus type 1 (HSV-1) HSZP interferes also after antibody neutralization with early shutoff of host protein synthesis induced by HSV-1 KOS

Summary The HSZP strain of herpes simplex virus type 1 (HSV-1) is defective with respect to the early shutoff of host protein synthesis. However, in superinfection experiments using Vero cells, the HSZP strain was effective, even after neutralization by antibody, at interfering with the early shutoff function of the HSV-1 KOS strain. Evidence was given that the observed interference was not due to exclusion of the KOS by HSZP at the level of adsorption or penetration. The neutralized KOS strain failed to induce early shutoff of host protein synthesis.     

Low infectivity of HSV-1 DNA caused by defective-interfering genomes

The infectivity of herpes simplex virus, type 1, strain ANG progeny DNA from standard virus infections and of progeny DNA from infections involving defective-interfering virus particles (DI DNA) was compared in transfection assays. No difference in infectivity of virus DNA isolated either from infected cells or from progeny virus was found for a given type of infection. However, the values for two types of infection differed markedly, with DI progeny DNA being less infectious by more than 2 log10. The low infectivity was mainly due to the presence of interfering DNA molecules in DI progeny DNA, regardless of whether intracellular DNA or DNA extracted from mature virions was analysed. The interfering capacity of DI progeny DNA did not depend on the integrity of the genomes. The physical proximity provided by simultaneous precipitation of infectious and of interfering DNA is an important factor influencing the degree to which DI DNA interferes. Interference by DI DNA in the transfection assay can be partly reversed by the addition of XbaI fragments of standard DNA; in control experiments this fragmented DNA was shown to lead to a reduction rather than to an enhancement of the infectivity of standard virus DNA.