Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of 4 respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.

Conserved Target for Group II Intron Insertion in Relaxase Genes of Conjugative Elements of Gram-Positive Bacteria

ABSTRACT The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.

Requirement of Varicella-Zoster Virus Immediate-Early 4 Protein for Viral Replication

ABSTRACT Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZV open reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.

Complement Regulation by Kaposi's Sarcoma-Associated Herpesvirus ORF4 Protein

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three types of human tumor: Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. The virus encodes a number of proteins that participate in disrupting the immune response, one of which was predicted by sequence analysis to be encoded by open reading frame 4 (ORF4). The predicted ORF4 protein shares homology with cellular proteins referred to as regulators of complement activation. In the present study, the transcription profile of the ORF4 gene was characterized, revealing that it encodes at least three transcripts, by alternative splicing mechanisms, and three protein isoforms. Functional studies revealed that each ORF4 protein isoform inhibits complement and retains a C-terminal transmembrane domain. Consistent with the complement-regulating activity, we propose to name the proteins encoded by the ORF4 gene collectively as KSHV complement control protein (KCP). KSHV ORF4 is the most complex alternatively spliced gene encoding a viral complement regulator described to date. KCP inhibits the complement component of the innate immune response, thereby possibly contributing to the in vivo persistence and pathogenesis of this virus.

Sequence of the 3′-terminal end (8·1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses

A cytopathogenic coronavirus, serologically identified as porcine haemagglutinating encephalomyelitis virus (HEV), has recently been associated with acute outbreaks of wasting and encephalitis in nursing piglets from pig farms in southern Québec and Ontario, Canada. The 3′-terminal end of the genome of the prototype HEV-67N strain and that of the recent Québec IAF-404 field isolate, both propagated in HRT-18 cells, were sequenced. Overall, sequencing data indicated that HEV has remained antigenically and genetically stable since its first isolation in North America in 1962. Compared with the prototype strain of bovine enteropathogenic coronavirus (BCoV), HEV, as well as the human respiratory coronavirus (HCoV-OC43) showed a major deletion in their ORF4 gene. Deduced amino acid sequences for both HEV strains revealed 89/88, 80, 93/92 and 95/94% identities with the structural proteins HE, S, M and N of BCoV and HCoV-OC43, respectively. Major variations were observed in the S1 portion of the S gene of both HEV strains, with only 73/71% amino acid identities compared with those of the two other haemagglutinating coronaviruses.

Cloning, Expression, and Sequence Analysis of the ORF4 Gene of the Porcine Reproductive and Respiratory Syndrome Virus MN-1b

Porcine reproductive and respiratory syndrome virus (PRRSV) MN-1b strain open reading frame 4 (ORF4) has been cloned, sequenced, and expressed in Escherichia coli. The homologies of nucleotide and amino acid sequences between MN-1b (US isolate) and LV (European isolate) are 69% and 64%, respectively. The data also showed that ORF4 of MN-1b is 36 bases shorter than that of LV. Western blot analysis of expressed recombinant ORF4 protein reacted with 65% (26/40) of PRRSV-infected pig sera tested. These results demonstrated that ORF4 of PRRSV may not be a well-conserved region.