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Myosin Va enhances secretion of herpes simplex virus 1 virions and cell surface expression of viral glycoproteins.

ABSTRACT: The final step in the egress of herpes simplex virus (HSV) virions requires virion-laden vesicles to bypass cortical actin and fuse with the plasma membrane, releasing virions into the extracellular space. Little is known about the host or viral proteins involved. In the current study, we noted that the conformation of myosin Va (myoVa), a protein known to be involved in melanosome and secretory granule trafficking to the plasma membrane in melanocytes and neuroendocrine cells, respectively, was altered by 4 h after infection with HSV-1 such that an N-terminal epitope expected to be masked in its inactive state was rendered immunoreactive. Wild-type myoVa localized throughout the cytoplasm and to a limited extent in the nuclei of HSV-infected cells. Two different dominant negative myoVa molecules containing cargo-binding domains but lacking the lever arms and actin-binding domains colocalized with markers of the trans-Golgi network (TGN). Expression of dominant negative myoVa isoforms reduced secretion of HSV-1 infectivity into the medium by 50 to 75%, reduced surface expression of glycoproteins B, M, and D, and increased intracellular virus infectivity to levels consistent with increased retention of virions in the cytoplasm. These data suggest that myoVa is activated during HSV-1 infection to help transport virion- and glycoprotein-laden vesicles from the TGN, through the cortical actin, to the plasma membrane. We cannot exclude a role for myoVa in promoting fusion of these vesicles with the inner surface of the plasma membrane. These data also indicate that myoVa is involved in exocytosis in human epithelial cells as well as other cell types.

SUBMITTER: Roberts KL

PROVIDER: S-EPMC2937760 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Myosin Va enhances secretion of herpes simplex virus 1 virions and cell surface expression of viral glycoproteins.

Roberts Kari L KL Baines Joel D JD

Journal of virology 20100714 19

The final step in the egress of herpes simplex virus (HSV) virions requires virion-laden vesicles to bypass cortical actin and fuse with the plasma membrane, releasing virions into the extracellular space. Little is known about the host or viral proteins involved. In the current study, we noted that the conformation of myosin Va (myoVa), a protein known to be involved in melanosome and secretory granule trafficking to the plasma membrane in melanocytes and neuroendocrine cells, respectively, was ...[more]

PMID: 20631136

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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:Herpesviruses-ubiquitous pathogens that cause persistent infections-have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact before receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form before fusion, interact at a steady level throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex before fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split-luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form receptor-independent complexes and interact at a steady level. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.

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Dataset Information

Myosin Va enhances secretion of herpes simplex virus 1 virions and cell surface expression of viral glycoproteins.

Publications

Myosin Va enhances secretion of herpes simplex virus 1 virions and cell surface expression of viral glycoproteins.

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