Dataset Information

RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.

ABSTRACT: The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts. These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression. We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time of assembly. To determine whether cis elements influenced RNA packaging, we constructed a recombinant HCMV mutant virus that expressed the yellow fluorescence protein (YFP) gene fused to the virion RNA UL21.5. We also constructed a mutant virus in which the UL21.5 transcription unit was replaced with the YFP gene. YFP RNA was incorporated into both viruses, indicating that RNA is incorporated in the absence of a virus-specific signal motif. Furthermore, with in situ hybridization, packaged transcripts were observed throughout the cytoplasm of the infected cells, including the site of virus assembly. Several proteins that nonspecifically interact with RNA, including the tegument protein pp28, were found within HCMV virions. These studies demonstrate that both viral and cellular RNAs are nonspecifically incorporated into HCMV, potentially through interactions with several virion proteins.

SUBMITTER: Terhune SS

PROVIDER: S-EPMC516422 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.

Terhune Scott S SS Schröer Jörg J Shenk Thomas T

Journal of virology 20041001 19

The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts. These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression. We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time ...[more]

PMID: 15367605

Similar Datasets

Project description:Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A)(+) RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs (U(L)10, U(L)34/U(L)35, U(L)36, U(L)42, U(L)48, U(L)51, U(S)1/U(S)1.5, U(S)8.5, and U(S)10/U(S)11) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the U(S)8 to U(S)12 genes yielded results similar to those described above, indicating that U(S)11, a known RNA binding protein, does not play a role in packaging RNA in virions. (iii) Cellular RNAs detected in virions were representative of the abundant cellular RNAs. Last, RNA extracted from virions was translated in vitro and the translation products were reacted with antibody to alphaTIF (VIP16). The immune precipitate contained a labeled protein with the apparent molecular weight of alphaTIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent selectivity in the packaging of the viral RNAs packaged in virions is unknown.

Project description:Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hot spots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild-type genomic RNAs. Interestingly, packaged DelVGs exhibited signs of enrichment for larger DelVGs suggesting that size is an important determinant of packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the influenza A virus replication machinery and shed light on the mechanisms that underly DelVG formation and packaging. IMPORTANCE Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes; hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This data set provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. In addition, by comparing the early intracellular DelVGs to those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild-type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.

Dataset Information

RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.

Publications

RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets