Gene mapping in Pichinde virus: assignment of viral polypeptides to genomic L and S RNAs.

Previous studies have demonstrated that Pichinde virus encodes at least three primary translation products. Using wild-type Pichinde and Munchique viruses and a reassortant between the two, designated RE-2, we were able to assign polypeptides L, GPC, and NP to viral L and S RNAs. The RE-2 virus contains the L RNA of Pichinde virus and the S RNA of Munchique virus. Two-dimensional tryptic peptide mapping of L-[35S]methionine-containing peptides demonstrated that NP and GPC were identical in Munchique and RE-2 viruses, and both differed from the corresponding Pichinde virus tryptic profiles. On the basis of this, NP and GPC must be encoded by viral S RNA. Similar comparisons for L polypeptide demonstrated that L is a virus-specific polypeptide encoded by L RNA.     

Strandedness of Pichinde virus RNA.

The Pichinde virus RNA did not possess the following characteristics of eucaryotic mRNA: polyadenylic acid sequence, capped methylated structure, and ability to direct protein synthesis in vitro. Polysomal RNA extracted from cells infected with Pichinde virus reannealed with 32P-labeled virus RNA, protecting about 60% of the latter against RNase degestion. The polyadenylic acid-containing polysomal RNA also reannealed to the 32P-labeled virus RNA to approximately the same extent. These indicate that the major part of the genomic RNA of Pichinde virus is negative stranded.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Photodynamic treatment of herpes simplex virus during its replicative cycle.

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation.     

Synthesis and turn-over of the replicative Cdc6 protein during the HeLa cell cycle.

The human replication protein Cdc6p is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle. However, the amounts of Cdc6p on chromatin remain high during S phase implying either that displaced Cdc6p can rebind to chromatin, or that Cdc6p is synthesized de novo. We have performed metabolic labeling experiments and determined that [35S]methionine is incorporated into Cdc6p at similar rates during the G1 phase and the S phase of the cell cycle. Newly synthesized Cdc6p associates with chromatin. Pulse-chase experiments show that chromatin-bound newly synthesized Cdc6p has a half life of 2-4 h. The results indicate that, once bound to chromatin, pulse-labeled new Cdc6p behaves just as old Cdc6p: it dissociates and eventually disappears from the nucleus. The data suggest a surprisingly dynamic behaviour of Cdc6p in the HeLa cell cycle.     

Differential accumulation of virus-specific RNA during the cell cycle of adenovirus-transformed rat embyro cells.

In adenovirus type 2-transformed rat embryo cells there is a threefold greater incorporation of [3-H]uridine into virus-specific RNA early in S phase than in late S or G2. This heightened accumulation of labeled RNA is true for both nuclear and cytoplasmic virus-specific labeling. Inhibition of DNA synthesis decreases the virus-specific RNA labeling, whereas reversal of inhibition again allows the elevated level of virus-specific RNA labeling.     

Analysis of polypeptides of virus-specific informosomes induced by tobacco mosaic virus and potato virus X

Informosome-like virus-specific ribonucleoprotein (vRNP) of tobacco mosaic virus (TMV) comprise a set of four major polypeptides having molecular weights of 17 500, 31 000, 37 000 and 39 000. Of the minor polypeptides, those of apparent molecular weights 25 000, 55 000, 68 000 and 70 000 had electrophoretic mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. Polypeptide with mol.wt. 175 000 is TMV coat protein so far as: a) vRNP was precipitated with immunoglobulins against TMV and TMV coat protein; b) it had electrophoretic mobility similar to mobility of TMV coat protein; c) the peptide map of polypeptides with mol.wts 31 000, 37 000 and 39 000 are probably virus-specific-products. This is supposed because they are not present in cell informosomes protein, and they are not revealed in vRNP induced in cells after infection with potato virus X (PVX). Electrophoresis of vRNP-PVX protein reveals polypeptides of 23 000 (PVX coat protein), 55 000, 70 000, 78 000, 95 000, 120 000 and 145 000.     

Sequential packaging of RNA genomic segments during the assembly of Bluetongue virus

Bluetongue virus (BTV), a member of the Orbivirus genus within the Reoviridae family, has a genome of 10 double-stranded RNA segments, with three distinct size classes. Although the packaging of the viral genome is evidently highly specific such that every virus particle contains a set of 10 RNA segments, the order and mechanism of packaging are not understood. In this study we have combined the use of a cell-free in vitro assembly system with a novel RNA–RNA interaction assay to investigate the mechanism of single-stranded (ss) RNAs packaging during nascent capsid assembly. Exclusion of single or multiple ssRNA segments in the packaging reaction or their addition in different order significantly altered the outcome and suggested a particular role for the smallest segment, S10. Our data suggests that genome packaging probably initiates with the smallest segment which triggers RNA–RNA interaction with other smaller segments forming a complex network. Subsequently, the medium to larger size ssRNAs are recruited until the complete genome is packaging into the capsid. The untranslated regions of the smallest RNA segment, S10, is critical for the instigation of this process. We suggest that the selective packaging observed in BTV may also apply to other members of the Reoviridae family.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

Characterization of polypeptides immunoprecipitable from Pichinde virus-infected BHK-21 cells

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

Sequence and structure of the catalytic RNA of hepatitis delta virus genomic RNA.

Human hepatitis delta virus (HDV) RNA has been shown to contain a self-catalyzed cleavage activity. The sequence requirement for its catalytic activity appears to be different from that of other known ribozymes. In this paper, we define the minimum contiguous sequence and secondary structure of the HDV genomic RNA required for the catalytic activity. By using nested-set deletion mutants, we have determined that the essential sequence for the catalytic activity is contained within no more than 85 nucleotides of HDV RNA. These results are in close agreement with the previous determinations and confirmed the relative insignificance of the sequence at the 5' side of the cleavage site. The smallest catalytic RNA, representing HDV genomic RNA nucleotide positions 683 to 770, was used as the basis for studying the secondary structure requirements for catalytic activity. Analysis of the RNA structure, using RNase V1, nuclease S1 and diethylpyrocarbonate treatments showed that this RNA contains at least two stem-and-loop structures. Other larger HDV RNA subfragments containing the catalytic activity also have a very similar secondary structure. By performing site-specific mutagenesis studies, it was shown that one of the stem-and-loop structures could be deleted to half of its original size without affecting the catalytic activity. In addition, the other stem-and-loop contained a six base-pair helix, and the structure, rather than the sequence, of this helix was required for the catalytic activity. However, the structure of a portion of the stem-and-loop remains uncertain. We also report that this RNA can be divided into two separate molecules, which alone did not have cleavage activity but, when mixed, one of the RNAs could be cleaved in trans. This study thus reveals some features of the secondary structure of the HDV genomic RNA involved in self-catalyzed cleavage. A model of this RNA structure is presented.     

Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length

There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virus-specific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virus-infected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virus-specific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.     

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.