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The tetraspanin protein CD9 modulates infection with human herpesvirus 6A and 6B in a CD46-dependent manner.

ABSTRACT: Tetraspanins are four-span transmembrane proteins that organize the membrane by forming tetraspanin-enriched microdomains. These have been shown to be important for virus entry. The human herpesvirus (HHV)-6A receptor CD46 is known to form complexes with the tetraspanin CD9 and β1-integrins, however the significance of this for HHV-6A infection remains unexplored. Using a genetic approach, we demonstrate that knock out of CD46 abolishes binding to and infection of SupT1 cells by both HHV-6A and HHV-6B, establishing CD46 as a necessary receptor for productive infection of these cells. Knock out of CD9 in SupT1 cells had no effect on binding of either virus to the cell surface, but it reduced expression of immediate early transcripts to between 25-60% compared with the wild type cells. Although HHV-6B required CD46 for infection of SupT1, infection of Molt3 cells was independent of CD46 expression. Conversely, the absence of CD9 expression promoted infection of Molt3 cells with HHV-6B, indicating a negative role of CD9 for CD46-independent infection. Taken together, these data demonstrate that CD9 modulates infection with HHV-6A/B by promoting CD46-dependent infection and impairing CD46-independent infection. This also suggests that HHV-6A is strictly dependent on CD46 for entry, although other proteins, like CD9, may enhance the infection, whereas HHV-6B is more promiscuous and may use CD134, as demonstrated by others, CD46 in SupT1, and a novel yet unidentified receptor in Molt3 cells.Importance The mechanisms of entry of human herpesvirus (HHV)-6A and HHV-6B into host cells are of significance in order to develop novel drugs that may inhibit infection. To elucidate the contribution of the membrane proteins CD9 and CD46, we employed a genetic approach that eliminated these molecules from the host cell. This demonstrated that CD46 is critical for infection by HHV-6A, whereas infection by HHV-6B appeared to be more promiscuous. The infection of a T-cell line in the absence of CD46 and CD134 strongly suggest that an additional receptor for HHV-6B entry exists. Moreover, elimination of CD9 and subsequent reconstitution experiments demonstrated that CD9 promoted infection with HHV-6A and HHV-6B mediated by CD46, but inhibited infection with HHV-6B that occurred independent of CD46. Together, this demonstrated a CD46-dependent role of CD9 during infection with HHV-6A and HHV-6B and emphasized that HHV-6B may employ different entry mechanisms in various cells.

SUBMITTER: Schack VR

PROVIDER: S-EPMC8103686 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:CD46 is a receptor for human herpesvirus 6A (HHV-6A) and is in some cells also important for infection with HHV-6B. CD46 has several isoforms of which the most commonly expressed can be distinguished by expression of a BC domain or a C domain in a serine-threonine-proline-rich (STP) extracellular region. Using a SupT1 CD46 CRISPR-Cas9 knockout model system reconstituted with specific CD46 isoforms, we demonstrated that HHV-6A infection was more efficient when BC isoforms were expressed as opposed to C isoforms, measured by higher levels of intracellular viral transcripts and recovery of more progeny virus. Although the B domain contains several O-glycosylations, mutations of Ser and Thr residues did not prevent infection with HHV-6A. The HHV-6A infection was blocked by inhibitors of clathrin-mediated endocytosis. In contrast, infection with HHV-6B was preferentially promoted by C isoforms mediating fusion-from-without, and this infection was less affected by inhibitors of clathrin-mediated endocytosis. Taken together, HHV-6A preferred BC isoforms, mediating endocytosis, whereas HHV-6B preferred C isoforms, mediating fusion-from-without. This demonstrates that the STP region of CD46 is important for regulating the mode of infection in SupT1 cells and suggests an epigenetic regulation of the host susceptibility to HHV-6A and HHV-6B infection. IMPORTANCE CD46 is the receptor used by human herpesvirus 6A (HHV-6A) during infection of T cells, but it is also involved in infection of certain T cells by HHV-6B. The gene for CD46 allows expression of several variants of CD46, known as isoforms, but whether the isoforms matter for infection of T cells is unknown. We used a genetic approach to delete CD46 from T cells and reconstituted them with separate isoforms to study them individually. We expressed the isoforms known as BC and C, which are distinguished by the potential inclusion of a B domain in the CD46 molecule. We demonstrate that HHV-6A prefers the BC isoform to infect T cells, and this occurs predominantly by clathrin-mediated endocytosis. In contrast, HHV-6B prefers the C isoform and infects predominantly by fusion-from-without. Thus, CD46 isoforms may affect susceptibility of T cells to infection with HHV-6A and HHV-6B.

Project description:Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.IMPORTANCE Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.

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The tetraspanin protein CD9 modulates infection with human herpesvirus 6A and 6B in a CD46-dependent manner.

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