Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions

The matrix (M1) protein of influenza A virus is a multifunctional protein that plays essential structural and functional roles in the virus life cycle. It drives virus budding and is the major protein component of the virion, where it forms an intermediate layer between the viral envelope and integral membrane proteins and the genomic ribonucleoproteins (RNPs). It also helps to control the intracellular trafficking of RNPs. These roles are mediated primarily via protein–protein interactions with viral and possibly cellular proteins. Here, the regions of M1 involved in binding the viral RNPs and in mediating homo-oligomerization are identified. In vitro, by using recombinant proteins, it was found that the middle domain of M1 was responsible for binding NP and that this interaction did not require RNA. Similarly, only M1 polypeptides containing the middle domain were able to bind to RNP–M1 complexes isolated from purified virus. When M1 self-association was examined, all three domains of the protein participated in homo-oligomerization although, again, the middle domain was dominant and self-associated efficiently in the absence of the N- and C-terminal domains. However, when the individual fragments of M1 were tagged with green fluorescent protein and expressed in virus-infected cells, microscopy of filamentous particles showed that only full-length M1 was incorporated into budding virions. It is concluded that the middle domain of M1 is primarily responsible for binding NP and self-association, but that additional interactions are required for efficient incorporation of M1 into virus particles

Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway

The mechanism of membrane scission during influenza A virus budding has been the subject of controversy. We confirm that influenza M1 binds VPS28, a subunit of the ESCRT-1 complex. However, confocal microscopy of infected cells showed no marked colocalisation between M1 and VPS28 or VPS4 ESCRT proteins, or relocalisation of the cellular proteins. Trafficking of HA and M1 appeared normal when endosomal sorting was impaired by expression of inactive VPS4. Overexpression of either isoform of VPS28 or wildtype or dominant negative VPS4 proteins did not alter production of filamentous virions. SiRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication. Furthermore, cells expressing wildtype or dominant-negative VPS4 replicated filamentous and non-filamentous strains of influenza to similar titres, indicating that influenza release is VPS4-independent. Overall, we see no role for the ESCRT pathway in influenza virus budding and the significance of the M1-VPS28 interaction remains to be determined. (C) 2009 Elsevier Inc. All rights reserved

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle

The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection

Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase

AbstractRespiratory syncytial virus RNA dependent RNA polymerase (RdRp) initiates RNA synthesis from the leader (le) and trailer-complement (trc) promoters. The RdRp can also add nucleotides to the 3′ end of the trc promoter by back-priming, but there is no evidence this occurs at the le promoter in infected cells. We examined how environmental factors and RNA sequence affect de novo RNA synthesis versus back-priming using an in vitro assay. We found that replacing Mg2+ with Mn2+ in the reaction buffer increased de novo initiation relative to back-priming, and different lengths of trc sequence were required for the two activities. Experiments with le RNA showed that back-priming occurred with this sequence in vitro, but less efficiently than with trc RNA. These findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay

The isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.

<p>(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (L_N812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-³²P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-³²P]GTP. Lane 2 is a control containing all four NTPs and [α-³²P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-³²P]GTP. In each panel, lane 1 shows the molecular weight ladder.</p

Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.

<p>(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ <u>CCG</u>CGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.</p

Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.

<p>(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-³²P]GTP, and lanes 4–6 show RNAs labeled with [α-³²P]ATP.</p