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

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

Initiation and regulation of paramyxovirus transcription and replication

AbstractThe paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication 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

Schematic diagrams illustrating the mechanisms of RSV transcription and replication initiation.

(A) Overview of the processes of transcription and replication, showing the capped and polyadenylated mRNAs and encapsidated antigenome and genome RNAs. The genes are shown as blue rectangles, with the gene start and gene end signals represented by white and black boxes. The le and tr promoter regions are indicated with green arrows. The le promoter yields mRNAs containing a methylguanosine cap (mG) and polyadenylate tail (An) and encapsidated antigenome; the tr promoter yields encapsidated genome RNA. The N protein that encapsidates the genome and antigenome RNA is shown as gray circles. Note that there is a gradient of transcription, which is not depicted here. (B) Initiation sites and RNAs produced from the 3ʹ end of the genome. The schematic shows the le region and the beginning of the first gene. The nucleotides in red are required for both transcription and replication, and are identical to the RSV L gs signal (CCCUGUUUUA). The NS1 gene is shown in a blue partial rectangle, with its gs signal shown in a white box. The initiation sites are shown with green arrows: those at 3C and the first gs signal are necessary for transcription; the initiation site at 1U is required for replication. The N protein is represented as a gray oval. It seems likely that if there were insufficient N protein available for encapsidation, RNA initiated at 1U would also be released after approximately 25 nt, allowing the polymerase to engage in transcription. (C) Model for initiation at two sites on the promoter. The L-P complex is represented with an orange oval. The polymerization active site, containing the NTP1 and NTP2 binding sites, is shown as a white box. The L-P complex could bind in two different registers on the promoter, with stability for one position or the other being conferred by the bound GTP, or ATP/CTP. The black dots indicate nucleotides that are repeated in the promoter sequence that could allow binding in two registers. le, leader; RSV, respiratory syncytial virus; tr, trailer.</p

Schematic diagram illustrating the relative levels of initiation from positions 1 and 3 of the <i>le</i> and <i>tr</i> promoters.

The genome and antigenome are shown as described in Fig 1A. The sequences of the le and tr promoters are shown in gray, with nucleotide differences shown in red. The green arrows show the initiation sites, with the weight of the arrows representing, approximately, the relative levels of initiation from each site. Note that the tr promoter also generates an approximately 25 nt RNA from position 3. The function of this RNA is not known, but it may be involved in subverting the cellular stress granule response [37]. le, leader; tr, trailer.</p

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

Effect of NTP concentration on RNA synthesis and 3′ nt addition.

<p>Reactions contained 0.2 µM TrC RNA with wt (lanes 2–4) or mutant (lane 5) RdRp, [α-³²P]GTP and varying concentrations of NTPs, from 200 µM to 1 mM of each NTP, as indicated. Lane 1 shows the molecular weight ladder.</p