A pre-immediate-early role for tegument ICP0 in the proteasome-dependent entry of herpes simplex virus

Herpes simplex virus (HSV) entry requires host cell 26S proteasomal degradation activity at a postpenetration step. When expressed in the infected cell, the HSV immediate-early protein ICP0 has E3 ubiquitin ligase activity and interacts with the proteasome. The cell is first exposed to ICP0 during viral entry, since ICP0 is a component of the inner tegument layer of the virion. The function of tegument ICP0 is unknown. Deletion of ICP0 or mutations in the N-terminal RING finger domain of ICP0 results in the absence of ICP0 from the tegument. We show here that these mutations negatively influenced the targeting of incoming capsids to the nucleus. Inhibitors of the chymotrypsin-like activity of the proteasome the blocked entry of virions containing tegument ICP0, including ICP0 mutants that are defective in USP7 binding. However, ICP0-deficient virions were not blocked by proteasomal inhibitors and entered cells via a proteasome-independent mechanism. ICP0 appeared to play a postpenetration role in cells that supported either endocytosis or nonendosomal entry pathways for HSV. The results suggest that ICP0 mutant virions are defective upstream of viral gene expression at a pre-immediate-early step in infection. We propose that proteasome-mediated degradation of a virion or host protein is regulated by ICP0 to allow efficient delivery of entering HSV capsids to the nuclear periphery.     

The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating.     

Herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic pathway

Herpes simplex virus (HSV) enters some laboratory cell lines via a pH-dependent, endocytic mechanism. We investigated whether this entry pathway is used in human cell types relevant to pathogenesis. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. In keratinocytes, incoming HSV particles colocalized with markers of endocytic uptake. Treatment with the isoflavone genistein, an inhibitor of protein tyrosine kinases, reduced the delivery of incoming viral particles to the nuclear periphery and virus-induced gene expression in keratinocytes but not neurons. Moreover, in keratinocyte monolayer islets, HSV infected both the inner and outer cells in a genistein-sensitive manner, suggesting viral endocytosis from both basolateral and apical plasma membrane surfaces. Together, the results indicate that HSV enters human epidermal keratinocytes, but not neurons, by a low-pH, endocytic pathway that is dependent on host tyrosine phosphorylation. Thus, HSV utilizes fundamentally different cellular entry pathways to infect important target cell populations.     

Proteolytic cleavage of VP1-2 is required for release of herpes simplex virus 1 DNA into the nucleus

In this report we propose a model in which after the herpes simplex virus (HSV) capsid docks at the nuclear pore, the tegument protein attached to the capsid must be cleaved by a serine or a cysteine protease in order for the DNA to be released into the nucleus. In support of the model are the following results. (i) Exposure of cells at the time of or before infection to l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK), a serine-cysteine protease inhibitor, prevents the release of viral DNA or expression of viral genes. TPCK does not block viral gene expression after entry of viral DNA into the nucleus. (ii) The tegument protein VP1-2, the product of the U(L)36 gene, is cleaved shortly after the entry of the HSV 1 (HSV-1) virion into the cell. (iii) The proteolytic cleavage of VP1-2 does not occur in cells that are infected with HSV-1 under conditions that prevent the release of the viral DNA into the nucleus. (iv) The proteolytic cleavage of VP1-2 occurs only after the capsid is attached to the nuclear pore. Thus, TPCK prevented the release of HSV-1 DNA into the nucleus when added to medium 1 hour after infection with tsB7 at 39.5 degrees C followed by a shift down to the permissive temperature. The ts lesion maps in the U(L)36 gene. At the nonpermissive temperature, the capsids accumulate at the nuclear pore but the DNA is not released into the nucleus.     

Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.     

Cellular and viral requirements for rapid endocytic entry of herpes simplex virus

It was recently demonstrated that herpes simplex virus (HSV) successfully infects Chinese hamster ovary (CHO) cells expressing glycoprotein D (gD) receptors and HeLa cells by an endocytic mechanism (A. V. Nicola, A. M. McEvoy, and S. E. Straus, J. Virol. 77:5324-5332, 2003). Here we define cellular and viral requirements of this pathway. Uptake of intact, enveloped HSV from the cell surface into endocytic vesicles was rapid (t(1/2) of 8 to 9 min) and independent of the known cell surface gD receptors. Following uptake from the surface, recovery of intracellular, infectious virions increased steadily up to 20 min postinfection (p.i.), which corresponds to accumulation of enveloped virus in intracellular compartments. There was a sharp decline in recovery by 30 min p.i., suggesting loss of the virus envelope as a result of capsid penetration from endocytic organelles into the cytosol. In the absence of gD receptors, endocytosed virions did not successfully penetrate into the cytosol but were instead transported to lysosomes for degradation. Inhibitors of phosphatidylinositol (PI) 3-kinase, such as wortmannin, blocked transport of incoming HSV to the nuclear periphery and virus-induced gene expression but had no effect on virus binding or uptake. This suggests a role for PI 3-kinase activity in trafficking of HSV through the cytosol. Viruses that lack viral glycoproteins gB, gD, or gH-gL were defective in transport to the nucleus and had reduced infectivity. Thus, similar to entry via direct penetration at the cell surface, HSV entry into cells by wortmannin-sensitive endocytosis is efficient, involves rapid cellular uptake of viral particles, and requires gB, gD, and gH-gL.     

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.     

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.     

Eclipse phase of herpes simplex virus type 1 infection: Efficient dynein-mediated capsid transport without the small capsid protein VP26

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-DeltaVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-DeltaVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-DeltaVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.     

Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.     

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.     

Function of dynein and dynactin in herpes simplex virus capsid transport

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.     

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Focal adhesion kinase plays a pivotal role in herpes simplex virus entry

Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore.     

Transportin 3 promotes a nuclear maturation step required for efficient HIV-1 integration

The HIV/AIDS pandemic is a major global health threat and understanding the detailed molecular mechanisms of HIV replication is critical for the development of novel therapeutics. To replicate, HIV-1 must access the nucleus of infected cells and integrate into host chromosomes, however little is known about the events occurring post-nuclear entry but before integration. Here we show that the karyopherin Transportin 3 (Tnp3) promotes HIV-1 integration in different cell types. Furthermore Tnp3 binds the viral capsid proteins and tRNAs incorporated into viral particles. Interaction between Tnp3, capsid and tRNAs is stronger in the presence of RanGTP, consistent with the possibility that Tnp3 is an export factor for these substrates. In agreement with this interpretation, we found that Tnp3 exports from the nuclei viral tRNAs in a RanGTP-dependent way. Tnp3 also binds and exports from the nuclei some species of cellular tRNAs with a defective 3'CCA end. Depletion of Tnp3 results in a re-distribution of HIV-1 capsid proteins between nucleus and cytoplasm however HIV-1 bearing the N74D mutation in capsid, which is insensitive to Tnp3 depletion, does not show nucleocytoplasmic redistribution of capsid proteins. We propose that Tnp3 promotes HIV-1 infection by displacing any capsid and tRNA that remain bound to the pre-integration complex after nuclear entry to facilitate integration. The results also provide evidence for a novel tRNA nucleocytoplasmic trafficking pathway in human cells.     

Crystal Structure of the Avian Astrovirus Capsid Spike

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.