Chemical crosslinking of proteins of the influenza virion. 1. Interrelationships.

Purified influenza virus (A/FPV/Rostock/34; H7N1) was reacted with one of three chemical crosslinking reagents [dimethylsuberimidate (DMS), tartryl diazide (TDA) and formaldehyde] under conditions designed to give a ladder of crosslinked polypeptides (putative homo- and heteropolymers) when analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The different virion polypeptides were identified by Western blotting with monospecific antisera against HA1, HA2, NP, and M1. When reacted with any crosslinker NP preferentially formed 2mer and 4mer homopolymers while M1 formed 2mers, 4mers, 6mers, and 8mers. 2mers and 3mers of HA1 were detected after crosslinking with TDA and DMS but homopolymers of HA2 could not be identified with certainty due to comigrating M1. One heteropolymer was clearly identified as 1NP:1M1 (with DMS and TDA) and others, as expected, as components of the haemagglutinin spike 1HA1:1HA2, 2HA1:2HA2, and 3HA1:3HA2. Formaldehyde gave rise only to HA1:HA2 polymers. The presence of other heteropolymers containing NP in conjunction with HA2 and HA1 seemed likely. Whenever HA2 ran with an Mr of about 50k it comigrated with M1 suggesting it may have formed (with DMS or TDA) a 1HA2:1M1 heterodimer. However it is possible that this band consisted of HA2 homodimers comigrating with M1 homodimers. Patterns of crosslinking with DMS and TDA were similar although not identical, but those obtained with formaldehyde were markedly different. All patterns were highly reproducible.