Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major subviral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins. Budding processes involve bud initiation, bud growth and bud release. Presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.
Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892 ) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a “Λ”-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a “Δ”-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies.
250) 1 The phosphoprotein pp150 is a structurally, immunogenically, and regulatorily important 2 capsid-associated tegument protein abundant in β-herpesviruses including 3 cytomegaloviruses (CMV), but absent in α-herpesviruses and γ-herpesviruses. In human 4 CMV (HCMV), bridging across each triplex and three adjacent major capsid proteins (MCPs) 5 is a group of three pp150 subunits in a "△"-shaped fortifying configuration, 320 of which 6 encase and stabilize the genome-containing capsid. Because murine CMV (MCMV) has been 7 used as a model for HCMV pathogenesis and therapeutic studies, one might expect that 8 pp150 and the capsid in MCMV and HCMV have similar structures. Here, by cryoEM and 9 sub-particle reconstructions, we have obtained structures of MCMV capsid and pp150 at near 10 atomic resolutions and built their atomic models. Surprisingly, the capsid-binding patterns of 11 pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In 12 MCMV, pp150 is absent on triplex Tc and exists as a "Λ"-shaped dimer on other triplexes, 13 leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of 14 three pp150 subunits encasing each HCMV capsid. Many more amino acids contribute to 15 pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible 16 thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is 17 essential in HCMV, pp150-deleted MCMV mutants remained viable though with attenuated 18 infectivity and exhibiting defects in retaining viral genome. These results support targeting 19 capsid proteins, but invalidate targeting pp150, when using MCMV as a model for HCMV 20 pathogenesis and therapeutic studies. 21 22 36 Keywords: cryoEM, β-herpesvirus, murine cytomegalovirus, human cytomegalovirus, 37 pp150, pUL32, pM32, bacterial artificial chromosome-based mutagenesis 38 4 | P a g e Recent high-resolution cryoEM structures of human herpesviruses (12-14), particularly 52the demonstration of inhibitors designed based on the structure of small capsid protein (SCP) 53 (12, 13), have opened the door to structure-guided design of new drugs and vaccines 54 targeting HCMV capsid proteins and the β-herpesvirus-specific tegument protein pUL32 (or 55 phosphoprotein pp150, see review 15). The cryoEM reconstruction of HCMV at 3.9 Å 56 resolution (14) reveals that pUL32 forms a unique capsid-binding tegument layer, likely to 57 secure encapsidation of its dsDNA genome of 235 kbp, which is the largest among all 58 herpesviruses. Particularly, 320 groups of three pUL32nt subunits form a "△"-shaped 59 fortifying structure on every triplex. pUL32 is an abundant and immunogenic protein that is 60 5 | P a g e essential for HCMV virion egress and maturation (16, 17). Yet careful examination of their 61 genomes suggests there might be structural differences between HCMV and MCMV. For 62 example, HCMV pUL32 sequence is about 40% longer than pM32 (the homolog of pUL32 in 63 MCMV) (18), suggesting that an examination of the structure ...
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